Δ4-desaturase genes and uses thereof

ABSTRACT

The subject invention relates to the identification of genes involved in the desaturation of polyunsaturated fatty acids at carbon 4 (i.e., “Δ4-desaturase”). In particular, Δ4-desaturase may be utilized, for example, in the conversion of adrenic acid to ω6-docosapentaenoic acid and in the conversion of ω3-docosapentaenoic acid to docosahexaenoic acid. The polyunsaturated fatty acids produced by use of the enzyme may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.

The subject application is a divisional of pending U.S. patentapplication Ser. No. 10/120,637, filed on Apr. 11, 2002, now U.S. Pat.No. 7,045,683 which is hereby incorporated in its entirety by reference.

The subject application is a continuation-in-part of U.S. patentapplication Ser. No. 09/849,199, filed on May 4, 2001, now abandonedhereby incorporated in its entirety by reference.

BACKGROUND OF THE INVENTION

1. Technical Field

The subject invention relates to the identification and isolation ofgenes that encode enzymes (e.g., Thraustochytrium aureum Δ4-desaturase,Schizochytrium aggregatum Δ4-desaturase and Isochrysis galbanaΔ4-desaturase) involved in the synthesis of polyunsaturated fatty acidsand to uses thereof. In particular, Δ4-desaturase catalyzes theconversion of, for example, adrenic acid (22:4n-6) toω6-docosapentaenoic acid (22:5n-6) and the conversion ofω3-docosapentaenoic acid (22:5n-3) to docosahexaenoic acid (22:6n-3).The converted products may then be utilized as substrates in theproduction of other polyunsaturated fatty acids (PUFAs). The product orother polyunsaturated fatty acids may be added to pharmaceuticalcompositions, nutritional composition, animal feeds as well as otherproducts such as cosmetics.

2. Background Information

Desaturases are critical in the production of long-chain polyunsaturatedfatty acids that have many important functions. For example,polyunsaturated fatty acids (PUFAs) are important components of theplasma membrane of a cell, where they are found in the form ofphospholipids. They also serve as precursors to mammalian prostacyclins,eicosanoids, leukotrienes and prostaglandins.

Additionally, PUFAs are necessary for the proper development of thedeveloping infant brain as well as for tissue formation and repair. Inview of the biological significance of PUFAs, attempts are being made toproduce them, as well as intermediates leading to their production, inan efficient manner.

A number of enzymes, most notably desaturase and elongases, are involvedin PUFA biosynthesis (see FIG. 1). For example, elongase (elo) catalyzesthe conversion of γ-linolenic acid (GLA) to dihomo-γ-linolenic acid(DGLA) and of stearidonic acid (18:4n-3) to (n-3)-eicosatetraenoic acid(20:4n-3). Linoleic acid (LA, 18:2n-9,12 or 18:2n-6) is produced fromoleic acid (18:1-Δ9) by a Δ12-desaturase. GLA (18:3n-6,9,12) is producedfrom linoleic acid by a Δ6-desaturase.

It must be noted that animals cannot desaturate beyond the Δ9 positionand therefore cannot convert oleic acid into linoleic acid. Likewise,γ-linolenic acid (ALA, 18:3n-9,12,15) cannot be synthesized by mammals.However, γ-linolenic acid can be converted to stearidonic acid (STA,18:4n-6,9,12,15) by a Δ6-desaturase (see PCT publication WO 96/13591 andThe FASEB Journal, Abstracts, Part I, Abstract 3093, page A532(Experimental Biology 98, San Francisco, Calif., Apr. 18–22, 1998); seealso U.S. Pat. No. 5,552,306), followed by elongation to(n-3)-eicosatetraenoic acid (20:4n-8,11,14,17) in mammals and algae.This polyunsaturated fatty acid (i.e., 20:4n-8,11,14,17) can then beconverted to eicosapentaenoic acid (EPA, 20:5n-5,8,11,14,17) by aΔ5-desaturase. EPA can then, in turn, be converted toω3-docosapentaenoic acid (22:5n-3) by an elongase. Isolation of anenzyme or its encoding gene, responsible for conversion ofω3-docosapentaenoic acid to docosahexaenoic acid (22:6n-3) has neverbeen reported. Two pathways for this conversion have been proposed (seeFIG. 1 and Sprecher, H., Curr. Opin. Clin. Nutr. Metab. Care, Vol. 2, p.135–138, 1999). One of them involves a single enzyme, a Δ4-desaturasesuch as that of the present invention. In the n-6 pathway, dietarylinoleic acid may be converted to adrenic acid through a series ofdesaturation and elongation steps in mammals (see FIG. 1). Production ofω6-docosapentaenoic acid from adrenic acid is postulated to be mediatedby the Δ6-desaturase discussed above.

Other eukaryotes, including fungi and plants, have enzymes whichdesaturate at carbon 12 (see PCT publication WO 94/11516 and U.S. Pat.No. 5,443,974) and carbon 15 (see PCT publication WO 93/11245). Themajor polyunsaturated fatty acids of animals therefore are eitherderived from diet and/or from desaturation and elongation of linoleicacid or γ-linolenic acid. In view of these difficulties, it is ofsignificant interest to isolate genes involved in PUFA synthesis fromspecies that naturally produce these fatty acids and to express thesegenes in a microbial, plant, or animal system which can be altered toprovide production of commercial quantities of one or more PUFAs.

In view of the above discussion, there is a definite need for theΔ4-desaturase enzyme, the respective genes encoding this enzyme, as wellas recombinant methods of producing this enzyme. Additionally, a needexists for oils containing levels of PUFAs beyond those naturallypresent as well as those enriched in novel PUFAs. Such oils can only bemade by isolation and expression of the Δ4-desaturase gene(s).

All U.S. patents and publications referred to herein are herebyincorporated in their entirety by reference.

SUMMARY OF THE INVENTION

The present invention includes an isolated nucleotide sequence orfragment thereof comprising or complementary to a nucleotide sequenceencoding a polypeptide having desaturase activity. The amino acidsequence of the polypeptide has at least 50% sequence identity to anamino acid sequence selected from the group consisting of SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:37, SEQ ID NO:46 andSEQ ID NO:55. Also, in particular, the present invention encompasses anisolated nucleotide sequence or fragment thereof comprising orcomplementary to a nucleotide sequence encoding a polypeptide havingdesaturase activity, wherein the amino acid sequence of said polypeptidehas at least 30% identity to the amino acid sequence of SEQ ID NO:55.

Additionally, the present invention encompasses an isolated nucleotidesequence or fragment thereof comprising or complementary to a nucleotidesequence having at least 50% sequence identity to a nucleotide sequenceselected from the group consisting of SEQ ID NO:14, SEQ ID NO:15 and SEQID NO:16, SEQ ID NO:17, SEQ ID NO:36, SEQ ID NO:45 and SEQ ID NO:54. Inparticular, the present invention includes an isolated nucleotidesequence or fragment thereof comprising or complementary to a nucleotidesequence having at least 40% identity to the nucleotide sequence of SEQID NO:54.

Each of the sequences described above encodes a functionally activedesaturase that utilizes a monounsaturated or polyunsaturated fatty acidas a substrate. The nucleotide sequences may be derived for example,from a fungus or an algae. In particular, when the nucleotide sequencecomprises SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17 orSEQ ID NO:45, may be derived, for example, from the fungusThraustochytrium aureum. The sequence comprising SEQ ID NO:36 may bederived, for example, from the fungus Schizochytrium aggregatum. Thesequence comprising SEQ ID NO:54 may be derived, for example, from thealgae Isochrysis galbana. The present invention also includes purifiedprotein and fragments thereof encoded by the above-referenced nucleotidesequences.

In particular, the present invention also includes a purifiedpolypeptide which desaturates polyunsaturated fatty acids at carbon 4and has an amino acid sequence having at least 50% identity to an aminoacid sequence selected from the group consisting of SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:37, SEQ ID NO:46 and SEQ IDNO:55. In particular, the present invention also includes a purifiedpolypeptide which desaturates polyunsaturated fatty acids at carbon 4and has an amino acid sequence having at least 30% identity to the aminoacid sequence of SEQ ID NO:55.

Additionally, the present invention includes a method of producing adesaturase comprising the steps of: isolating a nucleotide sequencecomprising or complementary to a nucleotide sequence encoding apolypeptide having an amino acid sequence having at least 50% identityto an amino acid sequence selected from the group consisting of SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:37, SEQ IDNO:46 and SEQ ID NO:55 (or at least 30% identity to the amino acidsequence of SEQ ID NO:55) or having at least 50% sequence identity to anucleotide sequence selected from the group consisting of SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:36, SEQ ID NO:45 andSEQ ID NO:54 (or having, in particular, at least 40% sequence identityto SEQ ID NO:54); constructing a vector comprising: i) the isolatednucleotide sequence operably linked to ii) a promoter; and introducingsaid vector into a host cell for a time and under conditions sufficientfor expression of the desaturase. The host cell may be, for example, aeukaryotic cell or a prokaryotic cell. In particular, the prokaryoticcell may be, for example, E. coli, cyanobacteria or B. subtilis. Theeukaryotic cell may be, for example, a mammalian cell, an insect cell, aplant cell or a fungal cell (e.g., a yeast cell such as Saccharomycescerevisiae, Saccharomyces carlsbergensis, Candida spp., Lipomycesstarkey, Yarrowia lipolytica, Kluyveromyces spp., Hansenula spp.,Trichoderma spp. or Pichia spp.).

Moreover, the present invention also includes a vector comprising: anisolated nucleotide sequence comprising or complementary to a nucleotidesequence encoding a polypeptide having an amino acid sequence having atleast 50% amino acid identity to an amino acid sequence selected fromthe group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:37, SEQ ID NO:46 and SEQ ID NO:55 (or, in particular,at least 30% amino acid identity to SEQ ID NO:55) or having at least 50%sequence identity to a nucleotide sequence selected from the groupconsisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:36, SEQ ID NO:45 and SEQ ID NO:54 (or, in particular, at least40% identity to SEQ ID NO:54), operably linked to a promoter. Theinvention also includes a host cell comprising this vector. The hostcell may be, for example, a eukaryotic cell or a prokaryotic cell.Suitable eukaryotic cells and prokaryotic cells are as defined above.

Moreover, the present invention also includes a plant cell, plant orplant tissue comprising the above vector, wherein expression of thenucleotide sequence of the vector results in production of apolyunsaturated fatty acid by the plant cell, plant or plant tissue. Thepolyunsaturated fatty acid may be, for example, selected from the groupconsisting of ω6-docosapentaenoic acid or docosahexaenoic acid. Theinvention also includes one or more plant oils or acids expressed by theabove plant cell, plant or plant tissue.

Additionally, the present invention also encompasses a transgenic plantcomprising the above vector, wherein expression of the nucleotidesequence of the vector results in production of a polyunsaturated fattyacid in seeds of the transgenic plant.

The present invention also includes a method (“first method”) forproducing a polyunsaturated fatty acid comprising the steps of:isolating a nucleotide sequence comprising or complementary to anucleotide sequence encoding a polypeptide having an amino acid sequencehaving at least 50% amino acid sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:37, SEQ ID NO:46 and SEQ IDNO:55 (and, in particular, at least 30% amino acid sequence identity toSEQ ID NO:55) or having at least 50% sequence identity to a nucleotidesequence selected from the group consisting of SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16 and SEQ ID NO:17, SEQ ID NO:36, SEQ ID NO:45, andSEQ ID NO:54 (and, in particular, at least 40% with respect to SEQ IDNO:54); constructing a vector comprising the isolated nucleotidesequence; introducing the vector into a host cell for a time and underconditions sufficient for expression of Δ4-desaturase; and exposing theexpressed Δ4-desaturase to a substrate polyunsaturated fatty acid inorder to convert the substrate to a product polyunsaturated fatty acid.The substrate polyunsaturated fatty acid may be, for example, adrenicacid or ω3-docospentaenoic acid, and the product polyunsaturated fattyacid may be, for example, ω6-docosapentaenoic acid or docosahexaenoicacid, respectively. This method may further comprise the step ofexposing the product polyunsaturated fatty acid to another enzyme (e.g.,a Δ4-desaturase, an elongase or another desaturase) in order to convertthe product polyunsaturated fatty acid to another polyunsaturated fattyacid (i.e., “second” method). In this method containing the additionalstep (i.e., “second” method), the product polyunsaturated fatty acid maybe, for example, ω6-docosapentaenoic acid, and the “another”polyunsaturated fatty acid may be docosahexaenoic acid.

Also, the present invention includes a method of producing apolyunsaturated fatty acid comprising the steps of: exposing a substratepolyunsaturated fatty acid to one or more enzymes selected from thegroup consisting of a desaturase and an elongase in order to convert thesubstrate to a product polyunsaturated fatty acid; and exposing theproduct polyunsaturated fatty acid to a Δ4-desaturase comprising anamino acid sequence selected from the group consisting of SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:37, SEQ ID NO:46 andSEQ ID NO:55, in order to convert the product polyunsaturated fatty acidto a final product polyunsaturated fatty acid.

For example, a substrate polyunsaturated fatty acid (e.g.,eicosapentaenoic acid) may be exposed to an elongase or desaturase(e.g., MELO4 or other elongases or desaturases of significance in thebiosynthetic pathway) in order to convert the substrate to a productpolyunsaturated fatty acid (e.g., ω3-docosapentaenoic acid). The productpolyunsaturated fatty acid may then be converted to a “final” productpolyunsaturated fatty acid (e.g., docosahexaenoic acid) by exposure tothe Δ4-desaturase of the present invention (see FIG. 1). Thus, theΔ4-desaturase is utilized in the last step of the method in order tocreate the “final” desired product. As another example, one may exposelinoleic acid to a Δ6-desaturase in order to create γ-linolenic acid(GLA), and then expose the GLA to an elongase and then to aΔ5-desaturase in order to create arachidonic acid (AA). The AA may thenbe exposed to an elongase in order to convert it to adrenic acid.Finally, the adrenic acid may be exposed to Δ4-desaturase in order toconvert it to ω6-docosapentaenoic acid (see FIG. 1). Thus, the methodinvolves the utilization of a linoleic acid substrate and a series ofdesaturase and elongase enzymes, in addition to the Δ4-desaturase, inorder to arrive at the final product. By use of a similar method, onemay also convert the substrate PUFA, γ-linolenic acid todocosoahexaenoic acid. Again, various desaturases and elongase are usedto ultimately arrive at ω3-docosapentaenoic acid which is then exposedto one or more of the Δ4-desaturases of the present invention in orderto convert it to docosahexaenoic acid. (Possible substrates includethose shown in FIG. 1, for example, linoleic acid, γ-linolenic acid,stearidonic acid, arachidonic acid, dihomo-γ-linolenic acid, adrenicacid, eicosapentaenoic acid and eicosatetraenoic acid.)

The present invention also encompasses a composition comprising at leastone polyunsaturated fatty acid selected from the group consisting of the“product” polyunsaturated fatty acid produced according to the methodsdescribed above and the “another” polyunsaturated fatty acid producedaccording to the methods described above. The product polyunsaturatedfatty acid may be, for example, ω6-docosapentaenoic acid ordocosahexaenoic acid. The another polyunsaturated fatty acid may be, forexample, docosahexaenoic acid.

Additionally, the present invention encompasses a method of preventingor treating a condition caused by insufficient intake of polyunsaturatedfatty acids comprising administering to the patient the compositionabove in an amount sufficient to effect prevention or treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the fatty acid biosynthetic pathway and the role ofΔ4-desaturase in this pathway.

FIG. 2 illustrates an amino acid comparison of Δ4-desaturases producedby four different plasmids (SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 andSEQ ID NO:21).

FIG. 3 illustrates the nucleotide sequence encoding Δ4-desaturase ofThraustochytrium aureum (ATCC 34304) from plasmid pRTA5 (SEQ ID NO:14).

FIG. 4 illustrates the nucleotide sequence encoding Δ4-desaturase ofThraustochytrium aureum (ATCC 34304) from plasmid pRTA6 (SEQ ID NO:15).

FIG. 5 illustrates the nucleotide sequence encoding Δ4-desaturase ofThraustochytrium aureum (ATCC 34304) from plasmid pRTA7 (SEQ ID NO:16).

FIG. 6 illustrates the nucleotide sequence encoding encodingΔ4-desaturase of Thraustochytrium aureum (ATCC 34304) from plasmid pRTA8(SEQ ID NO:17).

FIG. 7 illustrates the amino acid sequence of Δ4-desaturase ofThraustochytrium aureum (ATCC 34304) from plasmid pRTA5 (SEQ ID NO:18).

FIG. 8 illustrates the amino acid sequence of Δ4-desaturase ofThraustochytrium aureum (ATCC 34304) from plasmid pRTA6 (SEQ ID NO:19).

FIG. 9 illustrates the amino acid sequence of Δ4-desaturase ofThraustochytrium aureum (ATCC 34304) from plasmid pRTA7 (SEQ ID NO:20).

FIG. 10 illustrates the amino acid sequence of Δ4-desaturase ofThraustochytrium aureum (ATCC 34304) from plasmid pRTA8 (SEQ ID NO:21).

FIG. 11 illustrates the nucleotide and amino acid sequences describedherein.

FIG. 12 illustrates the amino acid sequence encoded by the elongase geneMELO4 from a mouse.

FIG. 13 illustrates the DNA sequence of the putative Δ4-desaturasessa.con (SEQ ID NO:24) generated from clones saa9 and saa5 from S.aggregatum (ATCC 28209) (see Example VI).

FIG. 14 illustrates the amino acid sequence (SEQ ID NO:25) of theputative Δ4-desaturase encoded by the ssa.con DNA sequence from S.aggregatum (ATCC 28209) (see Example VI).

FIG. 15 illustrates the alignment of the amino acids derived from thetranslation of the open reading frames of ssa.con DNA from S. aggregatum(ATCC 28209) (SEQ ID NO:25) and pRTA7 (SEQ ID NO:68) (see Example VI).

FIG. 16 illustrates the DNA sequence of the Δ4-desaturase from pRSA1(SEQ ID NO:36) S. aggregatum (ATCC 28209) (see Example VII).

FIG. 17 illustrates the amino acid sequence (SEQ ID NO:37) of theΔ4-desaturase encoded by the pRSA1 DNA sequence from S. aggregatum (ATCC28209) (see Example VII).

FIG. 18 illustrates the DNA sequence of the Δ4-desaturase from pRTA11(SEQ ID NO:45) T. aureum (BICC 7091) (see Example VII).

FIG. 19 illustrates the amino acid sequence (SEQ ID NO:46) of theputative Δ4-desaturase encoded by the pRTA11 DNA sequence from T. aureum(BICC 7091) (see Example VII).

FIG. 20 illustrates the DNA sequence of the Δ4-desaturase fromIsochrysis galbana (CCMP1323)(SEQ ID NO:54) present in clone pRIG6 (seeExample IX).

FIG. 21 illustrates the amino acid sequence (SEQ ID NO:55) of theΔ4-desaturase encoded by the pRIG6 DNA sequence from Isochrysis galbana(CCMP 1323) (see Example IX).

FIG. 22 illustrates the percent identity between the novel Δ4-desaturasefrom I. galbana (CCMP 1323) (SEQ ID NO:69) and the Δ4-desaturase fromThraustochytrium aureum (ATCC 34304) (SEQ ID NO:70).

DETAILED DESCRIPTION OF THE INVENTION

The subject invention relates to the nucleotide and translated aminoacid sequences of the Δ4-desaturase gene derived from the fungusThraustochytrium aureum (BICC 7091), the fungus Schizochytriumaggregatum, and the algae Isochrysis galbana. Furthermore, the subjectinvention also includes uses of these genes and of the enzymes encodedby these genes. For example, the genes and corresponding enzymes may beused in the production of polyunsaturated fatty acids such as, forinstance, ω6-docosapentaenoic acid and/or docosahexaenoic acid which maybe added to pharmaceutical compositions, nutritional compositions and toother valuable products.

The Δ4-Desaturase Genes and Enzymes Encoded Thereby

As noted above, the enzymes encoded by the Δ4-desaturase genes of thepresent invention are essential in the production of highly unsaturatedpolyunsaturated fatty acids having a length greater than 22 carbons. Thenucleotide sequences of the isolated Thraustochytrium aureumΔ4-desaturase genes, which differed based upon the plasmid created (seeExample III), are shown in FIGS. 3–6, and the amino acid sequences ofthe corresponding purified proteins are shown in FIG. 7-10. Anadditional, isolated T. aureum nucleotide sequence is shown in FIG. 18(see Example VII), and the encoded amino acid sequence is shown in FIG.19. The nucleotide sequences of the isolated Schizochytrium aggregatumΔ4-desaturase sequences are shown in FIGS. 13 and 16, and the encodedamino acid sequences are shown in FIGS. 14 and 17, respectively.Additionally, the nucleotide sequences of the isolated Isochrysisgalbana Δ4-desaturase sequence is shown in FIG. 20, and the amino acidsequence encoded by the nucleotide sequence is shown in FIG. 21.

As an example of the importance of the genes of the present invention,the isolated Δ4-desaturase genes convert adrenic acid toω6-docosapentaenoic acid or convert ω3-docosapentaenoic acid todocosahexaenoic acid.

It should be noted that the present invention also encompasses isolatednucleotide sequences (and the corresponding encoded proteins) havingsequences comprising, corresponding to, identical to, or complementaryto at least about 50%, preferably at least about 60%, and morepreferably at least about 70%, even more preferably at least about 80%,and most preferably at least about 90% sequence identity to SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17 (i.e., the nucleotidesequences of the Δ4-desaturase gene of Thraustochytrium aureum (ATCC34304)), to SEQ ID NO:36 (i.e., the nucleotide sequence of theΔ4-desaturase gene of Schizochytrium aggregatum (ATCC 28209) or to SEQID NO:45 (i.e., the nucleotide sequence of the Δ4-desaturase gene ofThraustochytrium aureum (BICC 7091)) or to SEQ ID NO:54 (i.e., thenucleotide sequence of the Δ4-desaturase gene of Isochrysis galabana),all described herein. With respect to the I. galbana sequence, inparticular, the present invention also encompasses nucleotide sequences(and the corresponding encoded proteins) having sequences comprising,corresponding to, identical to, or complementary to at least 40%, morepreferably at least 60%, even more preferably at least 80%, and mostpreferably at least 90% of the nucleotide sequence of SEQ ID NO:54. (Allintegers between 40% and 100% are also considered to be within the scopeof the present invention with respect to percent identity.) Suchsequences may be derived from any source, either isolated from a naturalsource, or produced via a semi-synthetic route, or synthesized de novo.In particular, such sequences may be isolated or derived from sourcesother than described in the examples (e.g., bacteria, fungus, algae, C.elegans, mouse or human).

Furthermore, the present invention also encompasses fragments andderivatives of the nucleotide sequences of the present invention (i.e.,SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:36,SEQ ID NO:45 or SEQ ID NO:54), as well as of the sequences derived fromother sources, and having the above-described complementarity, identityor correspondence. Functional equivalents of the above full lengthsequences and fragments (i.e., sequences having Δ4-desaturase activity,as appropriate) are also encompassed by the present invention.

For purposes of the present invention, a “fragment” is of a nucleotidesequence is defined as a contiguous sequence of approximately at least6, preferably at least about 8, more preferably at least about 10nucleotides, and even more preferably at least about 15 nucleotidescorresponding to a region of the specified nucleotide sequence.

The invention also includes a purified polypeptide which desaturatespolyunsaturated fatty acids at the carbon 4 position and has at leastabout 50% amino acid similarity or identity, preferably at least about60% amino acid similarity or identity, more preferably at least about70% amino acid similarity or identity, even more preferably at leastabout 80% amino acid similarity or identity and most preferably at least90% amino acid similarity or identity to the amino acid sequences (i.e.,SEQ ID NO:18 (shown in FIG. 7), SEQ ID NO:19 (shown in FIG. 8), SEQ IDNO:20 (shown in FIG. 9), SEQ ID NO:21 (shown in FIG. 10), SEQ ID NO:37(shown in FIG. 17), SEQ ID NO:46 (shown in FIG. 19) and SEQ ID NO:55(shown in FIG. 21) of the above-noted proteins which are, in turn,encoded by the above-described nucleotide sequences. In particular, withrespect to the amino acid sequence of the I. galbana Δ4-desaturase, thepresent invention encompasses includes a purified polypeptide whichdesaturates polyunsaturated fatty acids at the carbon 4 position and hasat least about 30% amino acid similarity or identity, preferably atleast about 50% amino acid similarity or identity, more preferably atleast about 70% amino acid similarity or identity and most preferably atleast about 90% amino acid similarity or identity to the amino acidsequence of SEQ ID NO:55 (i.e., the amino acid sequence of the I.galbana Δ4-desaturase shown in FIG. 21). (All integers between 30% and100% similarity or identity are also included within the scope of thepresent invention.)

The term “identity” refers to the relatedness of two sequences on anucleotide-by-nucleotide basis over a particular comparison window orsegment. Thus, identity is defined as the degree of sameness,correspondence or equivalence between the same strands (either sense orantisense) of two DNA segments (or two amino acid sequences).“Percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over a particular region, determining thenumber of positions at which the identical base or amino acid occurs inboth sequences in order to yield the number of matched positions,dividing the number of such positions by the total number of positionsin the segment being compared and multiplying the result by 100. Optimalalignment of sequences may be conducted by the algorithm of Smith &Waterman, Appl. Math. 2:482 (1981), by the algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the method of Pearson & Lipman,Proc. Natl. Acad. Sci. (USA) 85:2444 (1988) and by computer programswhich implement the relevant algorithms (e.g., Clustal Macaw PileupHiggins et al., CABIOS. 5L151–153 (1989)), FASTDB (Intelligenetics),BLAST (National Center for Biomedical Information; Altschul et al.,Nucleic Acids Research 25:3389–3402 (1997)), PILEUP (Genetics ComputerGroup, Madison, Wis.) or GAP, BESTFIT, FASTA and TFASTA (WisconsinGenetics Software Package Release 7.0, Genetics Computer Group, Madison,Wis.). (See U.S. Pat. No. 5,912,120.)

For purposes of the present invention, “complementarity is defined asthe degree of relatedness between two DNA segments. It is determined bymeasuring the ability of the sense strand of one DNA segment tohybridize with the antisense strand of the other DNA segment, underappropriate conditions, to form a double helix. A “complement” isdefined as a sequence which pairs to a given sequence based upon thecanonic base-pairing rules. For example, a sequence A-G-T in onenucleotide strand is “complementary” to T-C-A in the other strand.

In the double helix, adenine appears in one strand, thymine appears inthe other strand. Similarly, wherever guanine is found in one strand,cytosine is found in the other. The greater the relatedness between thenucleotide sequences of two DNA segments, the greater the ability toform hybrid duplexes between the strands of the two DNA segments.

“Similarity” between two amino acid sequences is defined as the presenceof a series of identical as well as conserved amino acid residues inboth sequences. The higher the degree of similarity between two aminoacid sequences, the higher the correspondence, sameness or equivalenceof the two sequences. (“Identity between two amino acid sequences isdefined as the presence of a series of exactly alike or invariant aminoacid residues in both sequences.) The definitions of “complementarity”,“identity” and “similarity” are well known to those of ordinary skill inthe art.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 amino acids, morepreferably at least 8 amino acids, and even more preferably at least 15amino acids from a polypeptide encoded by the nucleic acid sequence.

The present invention also encompasses an isolated nucleotide sequencewhich encodes PUFA desaturase activity and that is hybridizable, undermoderately stringent conditions, to a nucleic acid having a nucleotidesequence comprising or complementary to the nucleotide sequencesdescribed above. A nucleic acid molecule is “hybridizable” to anothernucleic acid molecule when a single-stranded form of the nucleic acidmolecule can anneal to the other nucleic acid molecule under theappropriate conditions of temperature and ionic strength (see Sambrooket al., “Molecular Cloning: A Laboratory Manual, Second Edition (1989),Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization. “Hybridization” requires that two nucleic acidscontain complementary sequences. However, depending on the stringency ofthe hybridization, mismatches between bases may occur. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation. Such variables are wellknown in the art. More specifically, the greater the degree ofsimilarity or homology between two nucleotide sequences, the greater thevalue of Tm for hybrids of nucleic acids having those sequences. Forhybrids of greater than 100 nucleotides in length, equations forcalculating Tm have been derived (see Sambrook et al., supra). Forhybridization with shorter nucleic acids, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook et al., supra).

As used herein, an “isolated nucleic acid fragment or sequence” is apolymer of RNA or DNA that is single- or double-stranded, optionallycontaining synthetic, non-natural or altered nucleotide bases. Anisolated nucleic acid fragment in the form of a polymer of DNA may becomprised of one or more segments of cDNA, genomic DNA or synthetic DNA.(A “fragment” of a specified polynucleotide refers to a polynucleotidesequence which comprises a contiguous sequence of approximately at leastabout 6 nucleotides, preferably at least about 8 nucleotides, morepreferably at least about 10 nucleotides, and even more preferably atleast about 15 nucleotides, and most preferable at least about 25nucleotides identical or complementary to a region of the specifiednucleotide sequence.) Nucleotides (usually found in their5′-monophosphate form) are referred to by their single letterdesignation as follows: “A” for adenylate or deoxyadenylate (for RNA orDNA, respectively), “C” for cytidylate or deoxycytidylate, “G” forguanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

The terms “fragment or subfragment that is functionally equivalent” and“functionally equivalent fragment or subfragment” are usedinterchangeably herein. These terms refer to a portion or subsequence ofan isolated nucleic acid fragment in which the ability to alter geneexpression or produce a certain phenotype is retained whether or not thefragment or subfragment encodes an active enzyme. For example, thefragment or subfragment can be used in the design of chimeric constructsto produce the desired phenotype in a transformed plant. Chimericconstructs can be designed for use in co-suppression or antisense bylinking a nucleic acid fragment or subfragment thereof, whether or notit encodes an active enzyme, in the appropriate orientation relative toa plant promoter sequence.

The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases does not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant invention such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the invention encompasses more than the specificexemplary sequences.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.

“Native gene” refers to a gene as found in nature with its ownregulatory sequences. In contrast, “chimeric construct” refers to acombination of nucleic acid fragments that are not normally foundtogether in nature. Accordingly, a chimeric construct may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than thatnormally found in nature. (The term “isolated” means that the sequenceis removed from its natural environment.)

A “foreign” gene refers to a gene not normally found in the hostorganism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric constructs. A “transgene” is a genethat has been introduced into the genome by a transformation procedure.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to, promoters, translation leader sequences, introns,and polyadenylation recognition sequences.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoter sequences canalso be located within the transcribed portions of genes, and/ordownstream of the transcribed sequences. Promoters may be derived intheir entirety from a native gene, or be composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. It is understood by those skilled in the artthat different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most host cell types at most times arecommonly referred to as “constitutive promoters”. New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro andGoldberg, (1989) Biochemistry of Plants 15:1–82. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity.

An “intron” is an intervening sequence in a gene that does not encode aportion of the protein sequence. Thus, such sequences are transcribedinto RNA but are then excised and are not translated. The term is alsoused for the excised RNA sequences. An “exon” is a portion of the genesequence that is transcribed and is found in the mature messenger RNAderived from the gene, but is not necessarily a part of the sequencethat encodes the final gene product.

The “translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D. (1995)Molecular Biotechnology 3:225).

The “3′ non-coding sequences” refer to DNA sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal isusually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., (1989) PlantCell 1:671–680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a DNA that is complementary to andsynthesized from a mRNA template using the enzyme reverse transcriptase.The cDNA can be single-stranded or converted into the double-strandedform using the Klenow fragment of DNA polymerase I. “Sense” RNA refersto RNA transcript that includes the mRNA and can be translated intoprotein within a cell or in vitro. “Antisense RNA” refers to an RNAtranscript that is complementary to all or part of a target primarytranscript or mRNA and that blocks the expression of a target gene (U.S.Pat. No. 5,107,065). The complementarity of an antisense RNA may be withany part of the specific gene transcript, i.e., at the 5′ non-codingsequence, 3′ non-coding sequence, introns, or the coding sequence.“Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNAthat may not be translated but yet has an effect on cellular processes.The terms “complement” and “reverse complement” are used interchangeablyherein with respect to mRNA transcripts, and are meant to define theantisense RNA of the message.

The term “endogenous RNA” refers to any RNA which is encoded by anynucleic acid sequence present in the genome of the host prior totransformation with the recombinant construct of the present invention,whether naturally-occurring or non-naturally occurring, i.e., introducedby recombinant means, mutagenesis, etc.

The term “non-naturally occurring” means artificial, not consistent withwhat is normally found in nature.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis regulated by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of regulating the expressionof that coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

The term “expression”, as used herein, refers to the production of afunctional end-product. Expression of a gene involves transcription ofthe gene and translation of the mRNA into a precursor or mature protein.“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.“Co-suppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of identical or substantiallysimilar foreign or endogenous genes (U.S. Pat. No. 5,231,020).

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and pro-peptidesstill present. Pre- and pro-peptides may be but are not limited tointracellular localization signals.

“Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, resulting in geneticallystable inheritance. In contrast, “transient transformation” refers tothe transfer of a nucleic acid fragment into the nucleus, orDNA-containing organelle, of a host organism resulting in geneexpression without integration or stable inheritance. Host organismscontaining the transformed nucleic acid fragments are referred to as“transgenic” organisms. The preferred method of cell transformation ofrice, corn and other monocots is the use of particle-accelerated or“gene gun” transformation technology (Klein et al., (1987) Nature(London) 327:70–73; U.S. Pat. No. 4,945,050), or anAgrobacterium-mediated method using an appropriate Ti plasmid containingthe transgene (Ishida Y. et al., 1996, Nature Biotech. 14:745–750). Theterm “transformation” as used herein refers to both stabletransformation and transient transformation.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

“PCR” or “Polymerase Chain Reaction” is a technique for the synthesis oflarge quantities of specific DNA segments, consists of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.).Typically, the double stranded DNA is heat denatured, the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps is referred to as a cycle.

Polymerase chain reaction (“PCR”) is a powerful technique used toamplify DNA millions of fold, by repeated replication of a template, ina short period of time. (Mullis et al, Cold Spring Harbor Symp. Quant.Biol. 51:263–273 (1986); Erlich et al, European Patent Application50,424; European Patent Application 84,796; European Patent Application258,017, European Patent Application 237,362; Mullis, European PatentApplication 201,184, Mullis et al U.S. Pat. No. 4,683,202; Erlich, U.S.Pat. No. 4,582,788; and Saiki et al, U.S. Pat. No. 4,683,194). Theprocess utilizes sets of specific in vitro synthesized oligonucleotidesto prime DNA synthesis. The design of the primers is dependent upon thesequences of DNA that are desired to be analyzed. The technique iscarried out through many cycles (usually 20–50) of melting the templateat high temperature, allowing the primers to anneal to complementarysequences within the template and then replicating the template with DNApolymerase.

The products of PCR reactions are analyzed by separation in agarose gelsfollowed by ethidium bromide staining and visualization with UVtransillumination. Alternatively, radioactive dNTPs can be added to thePCR in order to incorporate label into the products. In this case theproducts of PCR are visualized by exposure of the gel to x-ray film. Theadded advantage of radiolabeling PCR products is that the levels ofindividual amplification products can be quantitated.

The terms “recombinant construct”, “expression construct” and“recombinant expression construct” are used interchangeably herein.These terms refer to a functional unit of genetic material that can beinserted into the genome of a cell using standard methodology well knownto one skilled in the art. Such construct may be itself or may be usedin conjunction with a vector. If a vector is used then the choice ofvector is dependent upon the method that will be used to transform hostplants as is well known to those skilled in the art. For example, aplasmid vector can be used. The skilled artisan is well aware of thegenetic elements that must be present on the vector in order tosuccessfully transform, select and propagate host cells comprising anyof the isolated nucleic acid fragments of the invention. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al., (1985) EMBO J. 4:2411–2418; De Almeida et al., (1989) Mol. Gen.Genetics 218:78–86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

Production of the Δ4-Desaturase Enyzme

Once the gene encoding the Δ4-desaturase enzyme has been isolated, itmay then be introduced into either a prokaryotic or eukaryotic host cellthrough the use of a vector or construct. The vector, for example, abacteriophage, cosmid or plasmid, may comprise the nucleotide sequenceencoding the Δ4-desaturase enzyme, as well as any regulatory sequence(e.g., promoter) which is functional in the host cell and is able toelicit expression of the desaturase encoded by the nucleotide sequence.The regulatory sequence (e.g., promoter) is in operable association withor operably linked to the nucleotide sequence. (A promoter is said to be“operably linked” with a coding sequence if the promoter affectstranscription or expression of the coding sequence.) Suitable promotersinclude, for example, those from genes encoding alcohol dehydrogenase,glyceraldehyde-3-phosphate dehydrogenase, phosphoglucoisomerase,phosphoglycerate kinase, acid phosphatase, T7, TPI, lactase,metallothionein, cytomegalovirus immediate early, whey acidic protein,glucoamylase, and promoters activated in the presence of galactose, forexample, GAL1 and GAL10. Additionally, nucleotide sequences which encodeother proteins, oligosaccharides, lipids, etc. may also be includedwithin the vector as well as other regulatory sequences such as apolyadenylation signal (e.g., the poly-A signal of SV-40T-antigen,ovalalbumin or bovine growth hormone). The choice of sequences presentin the construct is dependent upon the desired expression products aswell as the nature of the host cell.

As noted above, once the vector has been constructed, it may then beintroduced into the host cell of choice by methods known to those ofordinary skill in the art including, for example, transfection,transformation and electroporation (see Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Vol. 1–3, ed. Sambrook et al., Cold Spring HarborLaboratory Press (1989)). The host cell is then cultured under suitableconditions permitting expression of the genes leading to the productionof the desired PUFA, which is then recovered and purified.

Examples of suitable prokaryotic host cells include, for example,bacteria such as Escherchia coli, Bacillus subtilis as well asCyanobacteria such as Spirulina spp. (i.e., blue-green algae). Examplesof suitable eukaryotic host cells include, for example, mammalian cells,plant cells, yeast cells such as Saccharomyces cerevisiae, Saccharomycescarlsbergensis, Lipomyces starkey, Candida spp. such as Yarrowia(Candida) lipolytica, Kluyveromyces spp., Pichia spp., Trichoderma spp.or Hansenula spp., or fungal cells such as filamentous fungal cells, forexample, Aspergillus, Neurospora and Penicillium. Preferably,Saccharomyces cerevisiae (baker's yeast) cells are utilized.

Expression in a host cell can be accomplished in a transient or stablefashion. Transient expression can occur from introduced constructs whichcontain expression signals functional in the host cell, but whichconstructs do not replicate and rarely integrate in the host cell, orwhen the host cell is not proliferating. Transient expression also canbe accomplished by inducing the activity of a regulatable promoteroperably linked to the gene of interest, although such inducible systemsfrequently exhibit a low basal level of expression. Stable expressioncan be achieved by introduction of a construct that can integrate intothe host genome or that autonomously replicates in the host cell. Stableexpression of the gene of interest can be selected through the use of aselectable marker located on or transfected with the expressionconstruct, followed by selection for cells expressing the marker. Whenstable expression results from integration, the site of the construct'sintegration can occur randomly within the host genome or can be targetedthrough the use of constructs containing regions of homology with thehost genome sufficient to target recombination with the host locus.Where constructs are targeted to an endogenous locus, all or some of thetranscriptional and translational regulatory regions can be provided bythe endogenous locus.

A transgenic mammal may also be used in order to express the enzyme ofinterest (i.e., Δ4-desaturase), and ultimately the PUFA(s) of interest.More specifically, once the above-described construct is created, it maybe inserted into the pronucleus of an embryo. The embryo may then beimplanted into a recipient female. Alternatively, a nuclear transfermethod could also be utilized (Schnieke et al., Science 278:2130–2133(1997)). Gestation and birth are then permitted (see, e.g., U.S. Pat.No. 5,750,176 and U.S. Pat. No. 5,700,671). Milk, tissue or other fluidsamples from the offspring should then contain altered levels of PUFAs,as compared to the levels normally found in the non-transgenic animal.Subsequent generations may be monitored for production of the altered orenhanced levels of PUFAs and thus incorporation of the gene encoding thedesired desaturase enzyme into their genomes. The mammal utilized as thehost may be selected from the group consisting of, for example, a mouse,a rat, a rabbit, a pig, a goat, a sheep, a horse and a cow. However, anymammal may be used provided it has the ability to incorporate DNAencoding the enzyme of interest into its genome.

For expression of a desaturase polypeptide, functional transcriptionaland translational initiation and termination regions are operably linkedto the DNA encoding the desaturase polypeptide. Transcriptional andtranslational initiation and termination regions are derived from avariety of nonexclusive sources, including the DNA to be expressed,genes known or suspected to be capable of expression in the desiredsystem, expression vectors, chemical synthesis, or from an endogenouslocus in a host cell. Expression in a plant tissue and/or plant partpresents certain efficiencies, particularly where the tissue or part isone which is harvested early, such as seed, leaves, fruits, flowers,roots, etc. Expression can be targeted to that location with the plantby utilizing specific regulatory sequence such as those of U.S. Pat.Nos. 5,463,174, 4,943,674, 5,106,739, 5,175,095, 5,420,034, 5,188,958,and 5,589,379. Alternatively, the expressed protein can be an enzymewhich produces a product which may be incorporated, either directly orupon further modifications, into a fluid fraction from the host plant.Expression of a desaturase gene, or antisense desaturase transcripts,can alter the levels of specific PUFAs, or derivatives thereof, found inplant parts and/or plant tissues. The desaturase polypeptide codingregion may be expressed either by itself or with other genes, in orderto produce tissues and/or plant parts containing higher proportions ofdesired PUFAs or in which the PUFA composition more closely resemblesthat of human breast milk (Prieto et al., PCT publication WO 95/24494).The termination region may be derived from the 3′ region of the genefrom which the initiation region was obtained or from a different gene.A large number of termination regions are known to and have been foundto be satisfactory in a variety of hosts from the same and differentgenera and species. The termination region usually is selected as amatter of convenience rather than because of any particular property.

As noted above, a plant (e.g., Glycine max (soybean) or Brassica napus(canola)) or plant tissue may also be utilized as a host or host cell,respectively, for expression of the desaturase enzyme which may, inturn, be utilized in the production of polyunsaturated fatty acids. Morespecifically, desired PUFAS can be expressed in seed. Methods ofisolating seed oils are known in the art. Thus, in addition to providinga source for PUFAs, seed oil components may be manipulated through theexpression of the desaturase gene, as well as perhaps other desaturasegenes and elongase genes, in order to provide seed oils that can beadded to nutritional compositions, pharmaceutical compositions, animalfeeds and cosmetics. Once again, a vector which comprises a DNA sequenceencoding the desaturase operably linked to a promoter, will beintroduced into the plant tissue or plant for a time and underconditions sufficient for expression of the desaturase gene. The vectormay also comprise one or more genes that encode other enzymes, forexample, Δ5-desaturase, elongase, Δ12-desaturase, Δ15-desaturase,Δ17-desaturase, and/or Δ19-desaturase. The plant tissue or plant mayproduce the relevant substrate (e.g., adrenic acid orω3-docosapentaenoic acid) upon which the enzyme acts or a vectorencoding enzymes which produce such substrates may be introduced intothe plant tissue, plant cell or plant. In addition, substrate may besprayed on plant tissues expressing the appropriate enzymes. Using thesevarious techniques, one may produce PUFAs (e.g., n-6 unsaturated fattyacids such as ω6-docosapentaenoic acid, or n-3 fatty acids such asdocosahexaenoic acid) by use of a plant cell, plant tissue or plant. Itshould also be noted that the invention also encompasses a transgenicplant comprising the above-described vector, wherein expression of thenucleotide sequence of the vector results in production of apolyunsaturated fatty acid in, for example, the seeds of the transgenicplant.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach and Weissbach, In: Methods for PlantMolecular Biology, (Eds.), Academic Press, Inc. San Diego, Calif.,(1988)). This regeneration and growth process typically includes thesteps of selection of transformed cells, culturing those individualizedcells through the usual stages of embryonic development through therooted plantlet stage. Transgenic embryos and seeds are similarlyregenerated. The resulting transgenic rooted shoots are thereafterplanted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a protein of interest is well known in theart. Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent invention containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135, U.S. Pat. No.5,518,908); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011,McCabe et. al., BiolTechnology 6:923 (1988), Christou et al., PlantPhysiol. 87:671–674 (1988)); Brassica (U.S. Pat. No. 5,463,174); peanut(Cheng et al., Plant Cell Rep. 15:653–657 (1996), McKently et al., PlantCell Rep. 14:699–703 (1995)); papaya; and pea (Grant et al., Plant CellRep. 15:254–258, (1995)).

Transformation of monocotyledons using electroporation, particlebombardment, and Agrobacterium have also been reported. Transformationand plant regeneration have been achieved in asparagus (Bytebier et al.,Proc. Natl. Acad. Sci. (USA) 84:5354, (1987)); barley (Wan and Lemaux,Plant Physiol 104:37 (1994)); Zea mays (Rhodes et al., Science 240:204(1988), Gordon-Kamm et al., Plant Cell 2:603–618 (1990), Fromm et al.,BiolTechnology 8:833 (1990), Koziel et al., BiolTechnology 11:194,(1993), Armstrong et al., Crop Science 35:550–557 (1995)); oat (Somerset al., BiolTechnology 10:1589 (1992)); orchard grass (Horn et al.,Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., TheorAppl. Genet.205:34 (1986); Part et al., Plant Mol. Biol. 32:1135–1148, (1996);Abedinia et al., Aust. J. Plant Physiol. 24:133–141 (1997); Zhang andWu, Theor. Appl. Genet. 76:835 (1988); Zhang et al. Plant Cell Rep.7:379, (1988); Battraw and Hall, Plant Sci. 86:191–202 (1992); Christouet al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992));tall fescue (Wang et al., BiolTechnology 10:691 (1992)), and wheat(Vasil et al., BiolTechnology 10:667 (1992); U.S. Pat. No. 5,631,152).

Assays for gene expression based on the transient expression of clonednucleic acid constructs have been developed by introducing the nucleicacid molecules into plant cells by polyethylene glycol treatment,electroporation, or particle bombardment (Marcotte et al., Nature335:454–457 (1988); Marcotte et al., Plant Cell 1:523–532 (1989);McCarty et al., Cell 66:895–905 (1991); Hattori et al., Genes Dev.6:609–618 (1992); Goff et al., EMBO J. 9:2517–2522 (1990)).

Transient expression systems may be used to functionally dissect geneconstructs (see generally, Maliga et al., Methods in Plant MolecularBiology, Cold Spring Harbor Press (1995)). It is understood that any ofthe nucleic acid molecules of the present invention can be introducedinto a plant cell in a permanent or transient manner in combination withother genetic elements such as vectors, promoters, enhancers etc.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolating ofclones, (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989); Maliga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995);Birren et al., Genome Analysis: Detecting Genes, 1, Cold Spring Harbor,N.Y. (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, ColdSpring Harbor, N.Y. (1998); Plant Molecular Biology: A LaboratoryManual, eds. Clark, Springer, New York (1997)).

The substrates which may be produced by the host cell either naturallyor transgenically, as well as the enzymes which may be encoded by DNAsequences present in the vector which is subsequently introduced intothe host cell, are shown in FIG. 1.

Uses of the Δ4-Desaturase Gene and Enzyme Encoded Thereby

As noted above, the isolated desaturase genes and the desaturase enzymesencoded thereby have many uses. For example, the gene and correspondingenzyme may be used indirectly or directly in the production ofpolyunsaturated fatty acids, for example, Δ4-desaturase may be used inthe production of ω6-docosapentaenoic acid or docosahexaenoic acid.“Directly” is meant to encompass the situation where the enzyme directlyconverts the acid to another acid, the latter of which is utilized in acomposition (e.g., the conversion of adrenic acid to ω6-docosapentaenoicacid). “Indirectly” is meant to encompass the situation where an acid isconverted to another acid (i.e., a pathway intermediate) by thedesaturase (e.g., adrenic acid to ω6-docosapentaenoic acid) and then thelatter acid is converted to another acid by use of a desaturase ornon-desaturase enzyme (e.g., ω6-docosapentaenoic acid to docosahexaenoicacid by Δ19-desaturase). Also, the present invention includes “indirect”situations in which the PUFA is first converted to anotherpolyunsaturated fatty acid by a non-Δ4-desaturase enzyme (for example,an elongase or another desaturase) and then converted to a final productvia Δ4-desaturase. For example, eicosapentaenoic acid may be convertedto ω3-docosapentaenoic acid by an elongase, and then converted todocosahexaenoic acid by a Δ4-desaturase. These polyunsaturated fattyacids (i.e., those produced either directly or indirectly by activity ofthe desaturase enzyme) may be added to, for example, nutritionalcompositions, pharmaceutical compositions, cosmetics, and animal feeds,all of which are encompassed by the present invention. These uses aredescribed, in detail, below.

Nutritional Compositions

The present invention includes nutritional compositions. Suchcompositions, for purposes of the present invention, include any food orpreparation for human consumption including for enteral or parenteralconsumption, which when taken into the body (a) serve to nourish orbuild up tissues or supply energy and/or (b) maintain, restore orsupport adequate nutritional status or metabolic function.

The nutritional composition of the present invention comprises at leastone oil or acid produced directly or indirectly by use of the desaturasegene, in accordance with the present invention, and may either be in asolid or liquid form. Additionally, the composition may include ediblemacronutrients, vitamins and minerals in amounts desired for aparticular use. The amount of such ingredients will vary depending onwhether the composition is intended for use with normal, healthyinfants, children or adults having specialized needs such as those whichaccompany certain metabolic conditions (e.g., metabolic disorders).

Examples of macronutrients which may be added to the composition includebut are not limited to edible fats, carbohydrates and proteins. Examplesof such edible fats include but are not limited to coconut oil, borageoil, fungal oil, black current oil, soy oil, and mono- and diglycerides.Examples of such carbohydrates include but are not limited to glucose,edible lactose and hydrolyzed search. Additionally, examples of proteinswhich may be utilized in the nutritional composition of the inventioninclude but are not limited to soy proteins, electrodialysed whey,electrodialysed skim milk, milk whey, or the hydrolysates of theseproteins.

With respect to vitamins and minerals, the following may be added to thenutritional compositions of the present invention: calcium, phosphorus,potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc,selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other suchvitamins and minerals may also be added.

The components utilized in the nutritional compositions of the presentinvention will be of semi-purified or purified origin. By semi-purifiedor purified is meant a material which has been prepared by purificationof a natural material or by synthesis.

Examples of nutritional compositions of the present invention includebut are not limited to infant formulas, dietary supplements (e.g., adultnutritional products and oil), dietary substitutes, and rehydrationcompositions. Nutritional compositions of particular interest includebut are not limited to those utilized for enteral and parenteralsupplementation for infants, specialist infant formulas, supplements forthe elderly, and supplements for those with gastrointestinaldifficulties and/or malabsorption.

The nutritional composition of the present invention may also be addedto food even when supplementation of the diet is not required. Forexample, the composition may be added to food of any type including butnot limited to margarines, modified butters, cheeses, milk, yogurt,chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats,fish and beverages.

In a preferred embodiment of the present invention, the nutritionalcomposition is an enteral nutritional product, more preferably, an adultor pediatric enteral nutritional product. This composition may beadministered to adults or children experiencing stress or havingspecialized needs due to chronic or acute disease states. Thecomposition may comprise, in addition to polyunsaturated fatty acidsproduced in accordance with the present invention, macronutrients,vitamins and minerals as described above. The macronutrients may bepresent in amounts equivalent to those present in human milk or on anenergy basis, i.e., on a per calorie basis.

Methods for formulating liquid or solid enteral and parenteralnutritional formulas are well known in the art. (See also the Examplesbelow.)

The enteral formula, for example, may be sterilized and subsequentlyutilized on a ready-to-feed (RTF) basis or stored in a concentratedliquid or powder. The powder can be prepared by spray drying the formulaprepared as indicated above, and reconstituting it by rehydrating theconcentrate. Adult and pediatric nutritional formulas are well known inthe art and are commercially available (e.g., SIMILAC®, ENSURE®, JEVITY®and ALIMENTUM® from Ross Products Division, Abbott Laboratories,Columbus, Ohio). An oil or acid produced in accordance with the presentinvention may be added to any of these formulas.

The energy density of the nutritional compositions of the presentinvention, when in liquid form, may range from about 0.6 Kcal to about 3Kcal per ml. When in solid or powdered form, the nutritional supplementsmay contain from about 1.2 to more than 9 Kcals per gram, preferablyabout 3 to 7 Kcals per gm. In general, the osmolality of a liquidproduct should be less than 700 mOsm and, more preferably, less than 660mOsm.

The nutritional formula may include macronutrients, vitamins, andminerals, as noted above, in addition to the PUFAs produced inaccordance with the present invention. The presence of these additionalcomponents helps the individual ingest the minimum daily requirements ofthese elements. In addition to the provision of PUFAs, it may also bedesirable to add zinc, copper, folic acid and antioxidants to thecomposition. It is believed that these substances boost a stressedimmune system and will therefore provide further benefits to theindividual receiving the composition. A pharmaceutical composition mayalso be supplemented with these elements.

In a more preferred embodiment, the nutritional composition comprises,in addition to antioxidants and at least one PUFA, a source ofcarbohydrate wherein at least 5 weight percent of the carbohydrate isindigestible oligosaccharide. In a more preferred embodiment, thenutritional composition additionally comprises protein, taurine, andcarnitine.

As noted above, the PUFAs produced in accordance with the presentinvention, or derivatives thereof, may be added to a dietary substituteor supplement, particularly an infant formula, for patients undergoingintravenous feeding or for preventing or treating malnutrition or otherconditions or disease states. As background, it should be noted thathuman breast milk has a fatty acid profile comprising from about 0.15%to about 0.36% as DHA, from about 0.03% to about 0.13% as EPA, fromabout 0.30% to about 0.88% as AA, from about 0.22% to about 0.67% asDGLA, and from about 0.27% to about 1.04% as GLA. Thus, fatty acids suchas AA, EPA and/or docosahexaenoic acid (DHA), produced in accordancewith the present invention, can be used to alter, for example, thecomposition of infant formulas in order to better replicate the PUFAcontent of human breast milk or to alter the presence of PUFAs normallyfound in a non-human mammal's milk. In particular, a composition for usein a pharmacologic or food supplement, particularly a breast milksubstitute or supplement, will preferably comprise one or more of AA,DGLA and GLA. More preferably, the oil will comprise from about C.3 to30% AA, from about 0.2 to 30% DGLA, and/or from about 0.2 to about 30%GLA.

Parenteral nutritional compositions comprising from about 2 to about 30weight percent fatty acids calculated as triglycerides are encompassedby the present invention. The preferred composition has about 1 to about25 weight percent of the total PUFA composition as GLA (U.S. Pat. No.5,196,198). Other vitamins, particularly fat-soluble vitamins such asvitamin A, D, E and L-carnitine can optionally be included. Whendesired, a preservative such as alpha-tocopherol may be added in anamount of about 0.1% by weight.

In addition, the ratios of AA, DGLA and GLA can be adapted for aparticular given end use. When formulated as a breast milk supplement orsubstitute, a composition which comprises one or more of AA, DGLA andGLA will be provided in a ratio of about 1:19:30 to about 6:1:0.2,respectively. For example, the breast milk of animals can vary in ratiosof AA:DGLA:GLA ranging from 1:19:30 to 6:1:0.2, which includesintermediate ratios which are preferably about 1:1:1, 1:2:1, 1:1:4. Whenproduced together in a host cell, adjusting the rate and percent ofconversion of a precursor substrate such as GLA and DGLA to AA can beused to precisely control the PUFA ratios. For example, a 5% to 10%conversion rate of DGLA to AA can be used to produce an AA to DGLA ratioof about 1:19, whereas a conversion rate of about 75% TO 80% can be usedto produce an AA to DGLA ratio of about 6:1. Therefore, whether in acell culture system or in a host animal, regulating the timing, extentand specificity of desaturase expression, as well as the expression ofother desaturases and-elongases, can be used to modulate PUFA levels andratios. The PUFAs produced in accordance with the present invention(e.g., AA and EPA) may then be combined with other PUFAs/acids (e.g.,GLA) in the desired concentrations and ratios.

Additionally, PUFA produced in accordance with the present invention orhost cells containing them may also be used as animal food supplementsto alter an animal's tissue or milk fatty acid composition to one moredesirable for human or animal consumption.

Pharmaceutical Compositions

The present invention also encompasses a pharmaceutical compositioncomprising one or more of the acids and/or resulting oils produced usingthe desaturase genes, in accordance with the methods described herein.More specifically, such a pharmaceutical composition may comprise one ormore of the acids and/or oils as well as a standard, well-known,non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle suchas, for example, phosphate buffered saline, water, ethanol, polyols,vegetable oils, a wetting agent or an emulsion such as a water/oilemulsion. The composition may be in either a liquid or solid form. Forexample, the composition may be in the form of a tablet, capsule,ingestible liquid or powder, injectible, or topical ointment or cream.Proper fluidity can be maintained, for example, by the maintenance ofthe required particle size in the case of dispersions and by the use ofsurfactants. It may also be desirable to include isotonic agents, forexample, sugars, sodium chloride and the like. Besides such inertdiluents, the composition can also include adjuvants, such as wettingagents, emulsifying and suspending agents, sweetening agents, flavoringagents and perfuming agents.

Suspensions, in addition to the active compounds, may comprisesuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanthor mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared usingtechniques well known in the art. For example, PUFAs produced inaccordance with the present invention can be tableted with conventionaltablet bases such as lactose, sucrose, and cornstarch in combinationwith binders such as acacia, cornstarch or gelatin, disintegratingagents such as potato starch or alginic acid, and a lubricant such asstearic acid or magnesium stearate. Capsules can be prepared byincorporating these excipients into a gelatin capsule along withantioxidants and the relevant PUFA(s). The antioxidant and PUFAcomponents should fit within the guidelines presented above.

For intravenous administration, the PUFAs produced in accordance withthe present invention or derivatives thereof may be incorporated intocommercial formulations such as Intralipids™. The typical normal adultplasma fatty acid profile comprises 6.64 to 9.46% of AA, 1.45 to 3.11%of DGLA, and 0.02 to 0.08% of GLA. These PUFAs or their metabolicprecursors can be administered alone or in combination with other PUFAsin order to achieve a normal fatty acid profile in a patient. Wheredesired, the individual components of the formulations may be providedindividually, in kit form, for single or multiple use. A typical dosageof a particular fatty acid is from 0.1 mg to 20 g (up to 100 g) dailyand is preferably from 10 mg to 1, 2, 5 or 10 g daily.

Possible routes of administration of the pharmaceutical compositions ofthe present invention include, for example, enteral (e.g., oral andrectal) and parenteral. For example, a liquid preparation may beadministered, for example, orally or rectally. Additionally, ahomogenous mixture can be completely dispersed in water, admixed understerile conditions with physiologically acceptable diluents,preservatives, buffers or propellants in order to form a spray orinhalant.

The route of administration will, of course, depend upon the desiredeffect. For example, if the composition is being utilized to treatrough, dry, or aging skin, to treat injured or burned skin, or to treatskin or hair affected by a disease or condition, it may perhaps beapplied topically.

The dosage of the composition to be administered to the patient may bedetermined by one of ordinary skill in the art and depends upon variousfactors such as weight of the patient, age of the patient, immune statusof the patient, etc.

With respect to form, the composition may be, for example, a solution, adispersion, a suspension, an emulsion or a sterile powder which is thenreconstituted.

The present invention also includes the treatment of various disordersby use of the pharmaceutical and/or nutritional compositions describedherein. In particular, the compositions of the present invention may beused to treat restenosis after angioplasty. Furthermore, symptoms ofinflammation, rheumatoid arthritis, asthma and psoriasis may also betreated with the compositions of the invention. Evidence also indicatesthat PUFAs may be involved in calcium metabolism; thus, the compositionsof the present invention may, perhaps, be utilized in the treatment orprevention of osteoporosis and of kidney or urinary tract stones.

Additionally, the compositions of the present invention may also be usedin the treatment of cancer. Malignant cells have been shown to havealtered fatty acid compositions. Addition of fatty acids has been shownto slow their growth, cause cell death and increase their susceptibilityto chemotherapeutic agents. Moreover, the compositions of the presentinvention may also be useful for treating cachexia associated withcancer.

The compositions of the present invention may also be used to treatdiabetes (see U.S. Pat. No. 4,826,877 and Horrobin et al., Am. J. Clin.Nutr. Vol. 57 (Suppl.) 732S–737S). Altered fatty acid metabolism andcomposition have been demonstrated in diabetic animals.

Furthermore, the compositions of the present invention, comprising PUFAsproduced either directly or indirectly through the use of the desaturaseenzymes, may also be used in the treatment of eczema, in the reductionof blood pressure, and in the improvement of mathematics examinationscores. Additionally, the compositions of the present invention may beused in inhibition of platelet aggregation, induction of vasodilation,reduction in cholesterol levels, inhibition of proliferation of vesselwall smooth muscle and fibrous tissue (Brenner et al., Adv. Exp. Med.Biol. Vol. 83, p.85–101, 1976), reduction or prevention ofgastrointestinal bleeding and other side effects of non-steroidalanti-inflammatory drugs (see U.S. Pat. No. 4,666,701), prevention ortreatment of endometriosis and premenstrual syndrome (see U.S. Pat. No.4,758,592), and treatment of myalgic encephalomyelitis and chronicfatigue after viral infections (see U.S. Pat. No. 5,116,871).

Further uses of the compositions of the present invention include use inthe treatment of AIDS, multiple sclerosis, and inflammatory skindisorders, as well as for maintenance of general health.

Additionally, the composition of the present invention may be utilizedfor cosmetic purposes. It may be added to pre-existing cosmeticcompositions such that a mixture is formed or may be used as a solecomposition.

Veterinary Applications

It should be noted that the above-described pharmaceutical andnutritional compositions may be utilized in connection with animals(i.e., domestic or non-domestic), as well as humans, as animalsexperience many of the same needs and conditions as humans. For example,the oil or acids of the present invention may be utilized in animal feedsupplements, animal feed substitutes, animal vitamins or in animaltopical ointments.

The present invention may be illustrated by the use of the followingnon-limiting examples:

EXAMPLE I Design of Degenerate Oligonucleotides for the Isolation ofDesaturases from Thraustochytrium aureum and cDNA Library Construction

The fatty acid composition analysis of the marine fungusThraustochytrium aureum (T. aureum) (ATCC 34304) was investigated todetermine the types and amounts of polyunsaturated fatty acids (PUFAs).This fungus had substantial amounts of longer chain PUFAs such asarachidonic acid (ARA, 20:4n-6) and eicosapentaenoic acid (EPA,20:5n-3). However, T. aureum also produced PUFAs such as adrenic acid(ADA, 22:4n-6), ω6-docosapentaenoic acid (ω6-DPA 22:5n-6),ω3-docosapentaenoic acid (ω3-DPA, 22:5n-3), with the highest amount offatty acid being docosahexaenoic acid (DHA, 22:6n-3) (see FIG. 1). Thusin addition to Δ6-, Δ5- and Δ17-desaturases, T. aureum probably containsa Δ19-desaturase which converts ADA to ω3-DPA or ω6-DPA to DHA and/or aΔ4-desaturase which desaturates ADA to ω6-DPA or ω3-DPA to DHA. The goalwas therefore to attempt to isolate the predicted desaturase genes fromT. aureum, and to verify the functionality of the enzymes by expressionin an alternate host.

To isolate genes encoding for functional desaturase enzymes, a cDNAlibrary was constructed. T aureum (ATCC 34304) cells were grown inBY+Media (#790, Difco, Detroit, Mich.) at room temperature for 4 days,in the presence of light, and with constant agitation (250 rpm) toobtain the maximum biomass. These cells were harvested by centrifugationat 5000 rpm for 10 minutes and rinsed in ice-cold RNase-free water.These cells were then lysed in a French press at 10,000 psi, and thelysed cells were directly collected into TB buffered phenol. Proteinsfrom the cell lysate were removed by repeated phenol: chloroform (1:1v/v) extraction, followed by a chloroform extraction. The nucleic acidsfrom the aqueous phase were precipitated at −70° C. for 30 minutes using0.3M (final concentration) sodium acetate (pH 5.6) and one volume ofisopropanol. The precipitated nucleic acids were collected bycentrifugation at 15,000 rpm for 30 minutes at 4° C., vacuum-dried for 5minutes and then treated with DNaseI (RNase-free) in 1×DNase buffer (20mM Tris-Cl, pH 8.0; 5mM MgCl₂) for 15 minutes at room temperature. Thereaction was quenched with 5 mM EDTA (pH 8.0) and the RNA furtherpurified using the Qiagen RiNeasy Maxi kit (Qiagen, Valencia, Calif.) asper the manufacturer's protocol.

Messenger RNA was isolated from total RNA using oligo dT celluloseresin, and the pBluescript II XR library construction kit (Stratagene,La Jolla, Calif.) was used to synthesize double stranded cDNA which wasthen directionally cloned (5′ EcoRI/3′ XhoI) into pBluescript II SK(+)vector (Stratagene, La Jolla, Calif.). The T. aureum library containedapproximately 2.5×10⁶ clones each with an average insert size ofapproximately 700 bp. Genomic DNA from PUFA-producing T. aureum cultureswas isolated by crushing the culture in liquid nitrogen and was purifiedusing Qiagen Genomic DNA Extraction Kit (Qiagen, Valencia, Calif.).

The approach taken was to design degenerate oligonucleotides (primers)that represent amino acid motifs that are conserved in knowndesaturases. These primers could be then used in a PCR reaction toidentify a fragment containing the conserved regions in the predicteddesaturase genes from fungi. Since the only fungal desaturases whichhave been identified are Δ5- and Δ6-desaturase genes from Mortierellaalpina (Genbank accession numbers AF067650, AB020032, respectively),desaturase sequences from plants as well as animals were taken intoconsideration during the design of these degenerate primers. Inparticular, known Δ5- and Δ6-desaturase sequences from the followingorganisms were used for the design of these degenerate primers:Mortierella alpina, Borago officinalis, Helianthus annuus, Brassicanapus, Dictyostelium discoideum, Rattus norvegicus, Mus musculus, Homosapiens, Caenorhabdftis elegans, Arab idopsis thaliana, and Ricinuscommunis. The degenerate primers used were as follows using the CODEHOPBlockmaker program

a. Protein motif 1: NH₃- VYDVTEWVKRHPGG -COOH (SEQ ID NO: 56) Primer RO834: 5′-GTBTAYGAYGTBACCGARTGGGTBAAGCGYC (SEQ ID NO: 1) AYCCBGGHGGH-3′ B.Protein Motif 2: NH₃- GASANWWKHQHNVHH -COOH (SEQ ID NO: 57) Primer RO835(Forward): 5-′GGHGCYTCCGCYAACTGGTGGAAGCAYCAGC (SEQ ID NO: 2)AYAACGTBCAYCAY-3′ Primer RO836 (Reverse):5-′RTGRTGVACGTTRTGCTGRTGCTTCCACCAG (SEQ ID NO: 3) TTRGCGGARGCDCC-3′ C.Protein Motif 3: NH₃- NYQIEHHLFPTM -COOH (SEQ ID NO: 58) Primer RO838(Reverse): 5′-TTGATRGTCTARCTYGTRGTRGASAARGGVT (SEQ ID NO: 4) GGTAC-3′In addition, two more primers were designed based on the 2nd and 3rdconserved ‘Histidine-box’ found in known

6-desaturases. These were:

Primer RO753 5′-CATCATCATXGGRAAXARRTGRTG-3′ (SEQ ID NO: 5) Primer RO7545′-CTACTACTACTACAYCAYACXTAYAC (SEQ ID NO: 6) XAAY-3′.The degeneracy code for the oligonucleotide sequences was: B=C,G,T;H=A,C,T; S=C,G; R=A,G; V=A,C,G; Y=C,T; D=A,T,C; X=A,C,G,T

EXAMPLE II Use of Degenerate Oligonucleotides for the Isolation of aDesaturase from a Fungus

To isolate putative desaturase genes, total RNA was isolated using thelithium chloride method (Hoge, et al. (1982) Exp. Mycol. 6:225–232).Approximately 5 μg was reverse transcribed using the SuperScriptPreamplification system (LifeTechnologies, Rockville, Md.) to producefirst strand cDNA. The following primer combinations were used:RO834/836, RO834/838, RO835/836, RO835/838 and RO753/754 were used inseveral PCR reactions with different thermocycling parameters and Taqpolymerase at annealing temperatures below 60° C., but no bands wereproduced.

In additional attempts to isolate fragments of desaturases, thedegenerate primers RO834/838 (designed with the block maker program) andRO753/754 were used in a 50 μl reaction. The following components werecombined: 2 μl of the first strand cDNA template, 20 mM Tris-HCl, pH8.4, 50 mM KCl, 1.5 mM MgCl₂, 200 μM each deoxyribonucleotidetriphosphate, 0.2 pmole final concentration of each primer and cDNApolymerase (Clonetech, Palo Alto, Calif.). Thermocycling was carried outas follows: an initial denaturation at 94° C. for 3 minutes, followed by35 cycles of; denaturation at 94° C. for 30 seconds, annealing at 60° C.for 30 seconds and extension at 72° C. for 1 minute. This was followedby a final extension at 72° C. for 7 minutes. Two faint bands ofapproximately 1000 bp were detected for primers RO834/838, while aslightly smaller but more intense band of 800–900 bp was found with theprimer pair RO753/754. The reactions were separated on a 1% agarose gel,excised, and purified with the QiaQuick Gel Extraction Kit (Qiagen,Valencia, Calif.). The staggered ends on these fragments were‘filled-in’ using T4 DNA polymerase (LifeTechnologies, Rockville, Md.)as per manufacture's specifications, and these DNA fragments were clonedinto the PCR-Blunt vector (Invitrogen, Carlsbad, Calif.). Therecombinant plasmids were transformed into TOP10 supercompetent cells(Invitrogen, Carlsbad, Calif.) and clones were partially sequenced.

Subsequently, the sequences of clone 30-3 (reaction with [RO834/838])and clone 17-1 (reaction with RO753/754) were found to overlap to createa 1313 bp fragment. The fragment was translated and Tfasta used tosearch the GenBank database. The highest match was Mortierella alpinaΔ5-desaturase (Genbank accession #AF067654) (27% homology in 202 aminoacids), Spirulina platensis Δ6-desaturase (Genbank accession numberX87094) (30% homology in 121 amino acids), Dictyostelium discoideumΔ5-desaturase (Genbank accession number AB02931) (26% homology in 131amino acids), and M. alpina Δ6-desaturase (accession number AF110510(30% homology in 86 amino acids). Since there was a reasonable degree ofamino acid homology to known desaturases, a full-length gene encoding apotential desaturase was sought to determine its activity when expressedin yeast.

EXAMPLE III Isolation of the Full Length Gene Sequence from T. aureum(ATCC 34304)

To find the full-length gene, two separate PCR reactions were carriedout in an attempt to determine the two ends of putative desaturase fromthe cDNA library. For the 3′ end of the gene, RO898 (SEQ ID NO:7)(5′-CCCAGTCACGACGTTGTAAAACGACGGCCAG-3′) (designed based on the sequenceof the pBluescript SK(+) vector (Stragene, La Jolla, Calif.) was used ina PCR amplification reaction along with a gene specific primer RO930(SEQ ID NO:8)(5′-GACGATTAACAAGGTGATTTCCCAGGATGTC). In this case, theAdvantage -GC cDNA PCR kit (Clonetech, Palo Alto, Calif.) was used toovercome PCR amplification problems that occur with GC rich sequences(61% for 1313 bp fragment). PCR thermocycling conditions were asfollows: the template was initially denatured at 94° C. for 3 minutes,followed by 30 cycles of [94° C. for 30 seconds, 52° C. for 30 seconds,and 72° C. for 1 minute], and finally an extension cycle at 72° C. for 7minutes with 20 pmoles of each primer. The PCR products thus obtainedwas resolved on a 1% agarose gel, excised, and gel purified using theQiagen Gel Extraction Kit (Qiagen, Valencia, Calif.). The staggered endson the fragment was ‘filled-in’ using T4 DNA polymerase(LifeTechnologies, Rockville, Md.) as per manufactures specificationsand cloned into the PCR-Blunt vector (Invitrogen, Carlsbad, Calif.) asdescribed in Example II. Clone 93-3 sequence overlapped the original1313 bp fragment and was found to contain an open reading frame, a stopcodon, and a poly A tail indicating that this was the 3′ end of thegene. Two primers were designed based on clone 93-3 sequence near thestop codon with an XhoI created site (underlined) as follows: RO973 (SEQID NO:9) (5′-GACTAACTCGAGTCACGTGGACCAGGCCGTGAGGTCCT-3′) and RO974 (SEQID NO:10) (5′-GACTAACTCGAGTTGACGAGGTTTGTAT GTTCGGCGGTTTGCTTG-3′). Twoprimers were deliberately chosen because RO973, that contained the stopcodon, was high in GC (60%) and might not amplify well. On the otherhand, RO974, downstream of the stop codon, was much lower in GC (48%).

Following the same protocol as described above to isolate the 5′ end ofthe gene, RO899 (SEQ ID NO:11) (5′-AGCGGATAACAATTTCACACAGGAAACAGC-3′)(designed based on the sequence of the pBluescript SK(+) vector) and thegene specific oligonucleotide RO1004 (SEQ ID NO:12)(5′-TGGCTACCGTCGTGCTGGATGCAAGTTCCG-3′) were used for amplification ofthe cDNA library. Amplification was carried out using 10 pmols of eachprimer and the cDNA polymerase kit (Clonetech, Palo Alto, Calif.). Thereaction conditions included an initial denaturation at 94° C. for 1minute, followed by 30 cycles of [94° C. for 30 seconds, 68° C. for 3minutes], and finally an extension cycle at 68° C. for 5 minutes. ThePCR products thus obtained were cloned into the PCR-Blunt vector(Invitrogen, Carlsbad, Calif.) following the same protocol as describedabove. The recombinant plasmids were transformed into TOP10supercompetent cells (Invitrogen, Carlsbad, Calif.), and clones weresequenced. Clone 1004-5 contained an open reading frame, several startcodons, and overlapped the original 1313 bp sequence indicating thatthis was the 5′ end of the gene.

To isolate the full-length gene, a primer for the 5′ end of the putativedesaturase was designed with a created EcoRI (underlined) as follows:RO1046 (SEQ ID NO:13) (5′-CGCATGGAATTCATGACGGTCGGGTTTGACGAAACGGTG-3′).

To isolate a full-length clone, both RO1046/973 and RO1046/974 were usedwith cDNA isolated from the library and genomic DNA as a target. BothcDNA polymerase (Clonetech, Palo Alto, Calif.) and -GC AdvantagePolymerase (Clonetech, Palo Alto, Calif.) were used to amplify theirrespective targets with 10 pmol of primer with the following reactionconditions: an initial denaturation at 94° C. for 1 minute, followed by30 cycles of [94° C. for 30 seconds, 68° C. for 3 minutes], and finallyan extension cycle at 68° C. for 5 minutes. The reactions were gelpurified, cut with EcoRI/XhoI and cloned into EcoRI/XhoI prepared yeastexpression vector pYX242 (Invitrogen, Carlsbad, Calif.) that had beentreated with shrimp alkaline phosphatase (Roche, Indianapolis, Ind.) toprevent recircularization. Initial analysis of the full-length sequencesshowed several base changes. Clones 112-3 and 112-5 (designated pRTA7and 8, respectively) were derived from the amplification with genomicDNA and -GC Advantage polymerase using primers RO1046/974. Clone 110-3(designated pRTA5) was derived from a reaction with RO1046/973, genomicDNA target and cDNA polymerase. Clone 111-1 (designated pRTA6) wasisolated from the reaction using RO1046/974, cDNA target and -GCAdvantage polymerase kit. The sequence of these four plasmids, pRTA5(SEQ ID NO:14), pRTA6 (SEQ ID NO:15), pRTA7 (SEQ ID NO:16), pRTA8 (SEQID NO:17) is shown in FIGS. 3–6, respectively. (Plasmids pRTA7 and pRTA8were deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110 on Apr. 19, 2001 and wereaccorded accession numbers PTA-3301 and PTA-3300, respectively.) Thisputative desaturase of 1548 bp and 515 amino acids (see FIGS. 7–10 andSEQ ID NOS:18, 19, 20, 21, respectively) had many of the characteristicsof described desaturases. The amino acids corresponding to the 5′ end ofthe enzyme are homologous to cytochrome b5. There are also number ofhistidine boxes at the following amino acids: 178-183-(Q)HDGSH (SEQ IDNO:59); 213-219-(Q)HVLGHH (SEQ ID NO:60); 262-265-HPWH (SEQ ID NO:61);271-275-HKFQH (SEQ ID NO:62); and 452-457(H)QIEHH (SEQ ID NO:63). Atleast either an H or a Q precedes three of these histidine boxes whichis unusual. Diclyostelium discoideum (Genbank accession number AB029311) has two similar boxes [(Q)HVIGHH (SEQ ID NO:64) and (H)QVVHH (SEQ IDNO:65)], while M. alpina (Genbank accession number AF067654) has(Q)HMLGHH (SEQ ID NO:66) and Synechocystis sp. only has one (H)QVTHH(SEQ ID 67).

The sequences of the various putative desaturases differed from eachother. Several of the base changes resulted in a change in amino acid,as shown in Table 1. These differences could be naturally occurringvariants, introduced by PCR mismatch during final amplification, or aPCR error when the initial cDNA was produced. There are 7 individualamino acid changes between the four plasmids, none of which are shared(see FIGS. 2A and B, underlined and bold amino acids). These differencescould alter the activity of the encoded enzyme.

TABLE 1 Amino Acid Differences in Different Clones Amino Acid NumberPRTA5 PRTA6 PRTA7 PRTA8 99 F S F F 280 F F L F 284 F F F S 317 Y Y N Y332 T M M M 410 T T T A 513 R W W W

EXAMPLE IV Expression of Plasmids Containing Putative Desaturases inYeast

All four plasmids were transformed into competent Saccharomycescerevisiae strain 334. Yeast transformation was carried out using theAlkali-Cation Yeast Transformation Kit (BIO 101, Vista, Calif.)according to conditions specified by the manufacturer. Transformantswere selected for leucine auxotrophy on media lacking leucine (DOB[-Leu]). To detect the specific desaturase activity of these clones,transformants were grown in the presence of 50 μM specific fatty acidsubstrates as listed below:

-   a. Linoleic acid (18:2n-6) (conversion to alpha-linolenic acid would    indicate Δ15-desaturase activity and conversion to gamma-linolenic    acid would indicate desaturase activity)-   b. Alpha-linolenic acid (18:3n-3) (conversion to stearidonic acid    would indicate Δ6-desaturase activity)-   c. Arachidonic acid (20:4n-6) (conversion to eicosapentaenoic acid    would indicate Δ17 desaturase activity).-   d. Adrenic acid (22:4n-6) (conversion to ω3-docosapentaenoic acid    would indicate Δ19-activity or conversion to ω6-docosapentaenoic    acid would indicate Δ4-desaturase activity.-   e. ω3-Docosapentaenoic acid (22:5:n-3) (conversion to    docosahexaenoic would indicate Δ4-desaturase activity    The negative control strain was S. cerevisiae 334 containing the    unaltered pYX242 vector, and these were grown simultaneously.

The cultures were vigorously agitated (250 rpm) and grown for 48 hours a24° C. in the presence of 50 μM (final concentration) of the varioussubstrates in 50 ml of media lacking leucine after inoculation withovernight growth of single colonies in yeast peptone dextrose broth(YPD) at 30° C. The cells were pelleted, and the pellets vortexed inmethanol; chloroform was added along with tritridecanoin (as an internalstandard). These mixtures were incubated for at least an hour at roomtemperature or at 4° C. overnight. The chloroform layer was extractedand filtered through a Whatman filter with 1 gm anhydrous sodium sulfateto remove particulates and residual water. The organic solvents wereevaporated at 40° C. under a stream of nitrogen. The extracted lipidswere then derivitized to fatty acid methyl esters (FAME) for gaschromatography analysis (GC) by adding 2 mls of 0.5 N potassiumhydroxide in methanol to a closed tube. The samples were heated to 95°C.–100° C. for 30 minutes and cooled to room temperature. Approximately2 ml of 14% borontrifluoride in methanol was added and the heatingrepeated. After the extracted lipid mixture cooled, 2 ml of water and 1ml of hexane were added to extract the fatty acid methyl esters (FAME)for analysis by GC. The percent conversion was calculated by dividingthe product produced by the sum of (the product produced+the substrateadded) and then multiplying by 100.

The results showed conversion of ω-3DPA to DHA and ADA to ω6-DPA. Thiswould indicate Δ4-desaturase activity (see Table 2).

TABLE 2 Percent Conversion of Different Substrate Concentrations toProduct 25 uM 50 uM 100 uM 25 uM 50 uM 100 uM Clone 22:4n − 6 22:4n − 622:4n − 6 22:5n − 3 22:5n − 3 22:5n − 3 PYX242 (control) 0 0 0 0 0 1.28PRTA5 3.91 0.9 1.24 10 6.89 3.1 PRTA6 4.69 2.77 1.18 14.26 8.52 4.98PRTA7 10.97 6.11 3.14 36.34 17.52 9.92 PRTA8 5.55 2.43 0.92 19.44 8.524.33 22:4n − 6 to 22:5n − 6 (Adrenic acid to ω6-Docosapentaenoic acid)22:5n − 3 to 22:6n − 3 (ω3-Docosapentaenoic acid to Docosahexaenoicacid)

In particular, this is the first demonstration of a Δ4-desaturase genewith in vivo expression data. The conversion for the four clones rangedfrom 3.9 1% to 10.97% for production of ω6-DPA from ADA and 10% to36.34% for production of DHA from ω-3DPA. The enzyme appears to be muchmore active in the production of DHA rather than ω6-DPA, as indicated bythe reduced percent conversion, 36.34% vs 10.97%, respectively, for 25μm of substrate for clone pRTA7. At 100 μm concentration of eithersubstrate, the percent conversion (see Table 2) as well as the amount ofproduct produced (data not shown) decreased, indicating that there maybe feedback inhibition of the desaturation step by the substrate. Theamount of ω3-DPA (22:5n-3) incorporated (as a percent of the totallipid) is similar for all four plasmids (see Table 3, below). Howeverthe amount produced as a percent of the total does vary from 2.74(pRTA5)to 8.11% (pRTA7). The difference in the conversion rates and percentproduced could be due to the difference in sequence, hence amino acidvariation of the encoded enzyme in the four plasmids.

TABLE 3 Fatty Acid as a Percentage of Total Lipid Extracted from Yeast22:4(n − 6) 22:5(n − 3) 22:5(n − 6) 22:6(n − 3) Clone IncorporatedProduced Incorporated Produced PYX242 38.96 0 11.2 0 (control) PRTA514.5 0.59 19.8 2.74 PRTA6 16.07 0.79 17.97 4.38 PRTA7 39.88 4.91 14.218.11 PRTA8 36.94 2.17 17.45 4.25 25 μM substrate data shown Key: 22:4(n− 6) = Adrenic acid 22:5(n − 3) = ω3-Docosapentaenoic acid 22:5(n − 6) =ω6-Docosapentaenoic acid 22:6(n − 3) = Docosahexaenoic acid

This data shows unequivocally that these plasmids indeed encode aΔ4-desaturase, which has preferred activity on conversion of ω3-DPA toDHA activity over conversion of ADA to ω6-DPA.

EXAMPLE V Expression of Δ4-Desaturase with the Mouse Elongase in Yeast

The plasmids pRTA7 and pRTA8 (which had the two highest percentconversion) may be individually co-transformed with pRMELO4, a clonethat contains a mouse elongase gene from pRAE-84 (see U.S. patentapplication Ser. No. 09/624,670 incorporated herein in its entirety byreference). The mouse elongase of 879 base pairs (see FIG. 12 and SEQ IDNO:22) may be cloned as an EcoRI/SalI fragment in the yeast expressionvector pYES2 (Invitrogen, Carlsbad, Calif.) at the EcoRI/XhoI sites.This elongase of 292 amino acids catalyzes several of the elongationsteps in the PUFA pathway, specifically AA to ADA and EPA to ω3-DPA. ADAand ω3-DPA are substrates for the Δ4-desaturase. Yeast transformants maybe selected on minimal media lacking leucine and uracil (DOB [-Leu-Ura])for selection of Δ4-desaturase (pRTA7 or pRTA8) and pRMELO4 (mouseelongase). Growth and expression of the yeast culture containing pRMELO4and pRTA7 or pRTA8 in minimal media lacking uracil and leucine and 2%galactose may result in elongation of exogenously added AA to ADA and Δ4desaturation to ω6-DPA. Additionally, supplementation of EPA to theyeast minimal media may result in elongation to ω3-DPA by the elongasewhich may then be desaturated by the Δ4-desaturase to produce DHA asshown in FIG. 1. This has been previously demonstrated with elongasesand other desaturases to produce AA and EPA (see PCT application WO00/12720) and provides parallel experimental data to show thatelongation of a substrate and subsequent desaturation can take place invivo in an organism such as yeast and potentially other organisms.Further, the present data demonstrates the ability of the Δ4-desaturaseto work with another enzyme in the PUFA biosynthetic pathway to produceeither ω6-DPA or DHA from the precursors AA and EPA, respectively.

EXAMPLE VI Homoloque of Δ4-Desaturase from Schizochytrium aggregatum(ATCC 28209)

In parallel to Example II, RNA was isolated by the acid phenol methodfrom Schizochytrium aggregatum (S. aggregatum) ATCC 28209. Briefly,pellets of S. aggregatum were washed with cold deionized water andrepelleted for 5 minutes at 3000 rpm. Approximatley 10 ml of TESsolution (10 mM Tris-CL pH 7.5, 10 mM EDTA, and 0.5% SDS) was used toresuspend the pellet. Then 10 ml of acid phenol was added and incubationfollowed for one hour at 65° C. The pellet was placed on ice for 5minutes, centrifuged for 5 minutes at 1000×g at 4° C., and the aqueousphase transferred to a new tube. An additional 10 ml of acid phenol wasadded to the aqueous phase, the mixture vortexed and separated asbefore. The aqueous phase containing the nucleic acids was transferredto a new tube. Approximately 1 ml of sodium acetate pH 5.3 and 25 ml ofice-cold ethanol were added for overnight precipitation at −70 C. Thenext day, the tubes were centrifuged for 15 minutes at 14,000 rpm at 4°C. and the supernatant decanted. The pellet was washed with 10 ml of 70%ethanol and centrifuged as in the previous step. The pellet was driedand resuspended in 500 ul of RNAse free deionized water. The RNA wasfurther purified using the Qiagen RNeasy Maxikit (Qiagen, Valencia,Calif.) as per the manufacturer's protocol.

cDNA was generated using oligo dT with the SuperScript Preamplificationsystem (Life Technologies, Rockville, Md.) with 5 ug of RNA from S.aggregatum. Since S. aggregatum produces large quantities of DHA, aΔ4-desaturase would be required for DHA production. In an identicalexperiment, primers RO753 (SEQ ID NO:5) and RO754 (SEQ ID NO:6) wereused in the same reaction as in Example II to produce a band around 800base pairs. As before the DNA generated from the PCR reaction wasseparated on a 1% gel, excised, purified, and cloned into the PCR-Bluntvector (Invitrogen, Carlsbad, Calif.). The DNA sequence generated fromclones saa9 and saa5 overlapped to create the sequence saa.con (SEQ IDNO:24 and FIG. 13). The translation of the open reading frame of saa.conDNA sequence to an amino acid sequence (SEQ ID NO:25 and FIG. 14)aligned with pRTA7 is shown in FIG. 15. The amino acid sequence of theΔ4-desaturase from clone pRTA7 has 79.1% identity with the translatedsaa.con sequence over 249 amino acids. This sequence, due to its highidentity with a known Δ4-desaturase, is most likely a fragment of aΔ4-desaturase from S. aggregatum. This example provides evidence thatthis procedure can be used to isolate Δ4-desaturases from otherorganisms.

EXAMPLE VII Isolation of Δ4-Desaturase Nucleotide Sequences fromSchizochytrium aggregatum (ATCC 28209) and Thraustochytrium aureum (BICC7091)

To isolate the 5′ and 3′-ends, new primers were designed based on theinternal sequence of the isolated S. aggregatum fragment shown inExample VI. For the 5 prime end of the gene, RO1240 (SEQ ID NO:26)(5′-CCC TCG ATG ATG TGG TTG ACG ATG AAC-3′) was used and subsequently 5prime nested primer RO1239 (SEQ ID NO:27) (5′-CGG AGC ATG GGG TAG GTGCTG AAG AC-3′). For the 3 prime end, RO1236 (SEQ ID NO:28) (5′-CCA ACTGCC GTT ACG CCA GCA AGT-3′) was used followed by 3 prime nested primerRO1237 (SEQ ID NO:29) (5′-CAA GCT CTT CTT CAT CGC CCA CTT TTC G-3′), fora second reaction to isolate the other end of the gene. RACE (rapidamplification of cDNA ends) ready cDNA was used as a target for thereactions. To prepare this material, approximately 5 μg of total RNA wasused according the manufacturer's direction with the GENRACER™ kit(Invitrogen, Carlsbad, Calif.) and SUPERSCRIPT II™ enzyme (Invitrogen,Carlsbad, Calif.) for reverse transcription to produce cDNA target. Forthe initial amplification of the ends, the following thermocyclingprotocol was used in a Perkin Elmer 9600: initial melt at 94° C. for 2minutes followed by 5 cycles of 94° C. for 30 seconds and 72° C. for 3minutes, 10 cycles of 94° C. 30 seconds, 70° C. for 30 seconds, and 72°C. for 3 minutes and 20 cycles of 94° C. for 30 seconds, 68° C. for 30seconds and 72° C. for 3 minutes, followed by an extension of 72° C. for10 minutes. The first PCR reaction was performed with 10 pMol of RO1240and 30 pMol GeneRacer™ 5 prime primer (SEQ ID NO:30) (5′-CGA CTG GAG CACGAG GAC ACT GA-3′) or RO1236 and GeneRacer™ 3 prime primer (SEQ IDNO:31) (5′-GCT GTC AAC GAT ACG CTA CGT AAC G-3′). The reaction contained1 ul of cDNA in a final volume of 50 ul with Platimum Taq™ PCRx(Clonetech, Palo Alto, Calif.) using MgSO₄ according to themanufacturer's directions. A nested reaction was performed with 1 ul ofthe initial reaction, 10 pmol of nested primer RO239 and 30 pmol of theGENRACER™ nested 5 prime primer (SEQ ID NO:32) (5′-GGA CAC TGA CAT GGACTG AAG GAG TA-3′) or GeneRacer™ nested 3 prime primer (SEQ ID NO:33)(5′-CGC TAC GTA ACG GCA TGA CAG TG-3′) and nested primer RO1237 usingthe same conditions as the first reaction. Agarose gel analysis of thePCR products showed a band around 800 base pairs for the 5 primereaction and approximately 600 base pairs for the 3 prime reaction.Subsequent cloning into pCR Blunt (Invitrogen, Carlsbad, Calif.),transformation into Top10 competent cells (Invitrogen, Carlsbad,Calif.), and sequencing revealed an open reading frame with both a startand stop codon. Primers RO1241 (SEQ ID NO:34) (5′-GAT ATC GAA TTC ATGACG GTG GGC GGC GAT GAG G-3′) and RO1242 (SEQ ID NO:35) (5′-GTA CTT AAGCTT TCA CTT GGA CTT GGG GTG GTC C-3′) with restrictions sites added forcloning (see underlined EcoRI, and HindIII respectively) were used toisolate a full length gene. As shown above, 10 pmol of primers RO1241and 1242 were used with Platimum TAQ™ PCRx (Clonetech, Palo Alto,Calif.) using MgSO₄ according to the manufacturer's protocol with 2 ulof the cDNA as target. The thermocycling parameters were as follows:initial melt at 94° C. for 2 minutes followed by 5 cycles of 94° C. for30 seconds and 72° C. for 2 minutes, 5 cycles of 94° C. 30 seconds, 70°C. for 2 minutes and 20 cycles of 94° C. for 30 seconds, 65° C. for 30seconds and 68° C. for 2 minutes, followed by an extension of 68° C. for10 minutes. The large product of the reaction was gel purified using theQIAQUICK gel purification kit (Qiagen, Valencia, Calif.) cut with EcoRIand HindIII and ligated to pYX242 EcoRI/HindIII linearized DNA with theRapid ligation kit (Roche, Indianapolis, Ind.) and designated pRSA-1.The clone pRSA1 contained a full length gene of 1530 bp (SEQ ID NO:36,FIG. 16) and an open reading frame of 509 amino acids (SEQ IN NO:37,FIG. 17). (Plasmid pRSA-1 was deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110 on Mar. 27,2002 and was accorded accession number PTA-4186.)

The second Δ4-desaturase was identified by a partial sequence isolatedusing the primer combination of RO1201 (SEQ ID NO:38) (5′-CGT GTT CGCTGC CTT TGT CGG AAC TTG CAT CC-3′ and RO1202 (SEQ ID NO:39) (5′-TTG ACAATA AAC ATG GAG GCG AGG ACC TCT CCG-3′) based on the sequence of pRTA7(SEQ IN NO:16) as described in Example III. The genomic DNA (gDNA), wasprepared as described in Example I, from Thraustochytrium aureum (BICC7091) (Biocon India Ltd., Bangalore, India). PCR amplification wascarried out in a 100 ul volume containing: 5 μl of isolated T7091 gDNA,1.0 U of cDNA Polymerase (Clonetech, Palo Alto, Calif.) and 10 pMol ofprimers according the manufacturer's protocol. Thermocycler conditionsin Perkin Elmer 9600 were as follows: 94° C. for 3 min, then 35 cyclesof 94° C. for 30 sec., 60° C. for 30 sec., and 72° C. for 1 min. PCR wasfollowed by an additional extension at 72° C. for 7 minutes. A 600 bpfragment was gel purified, ends filled-in using T4 DNA Polymerase(LifeTechnologies, Rockville, Md., cloned into the pCR-Blunt vector(Invitrogen, Co., Carlsbad, Calif.), and the recombinant plasmidstransformed into TOP10 supercompetent cells (Invitrogen, Carlsbad,Calif.). Sequencing of the clones revealed high homology with pRTA7(82.1% in 196 amino acids).

For isolation of a full-length gene, a cDNA library was constructed withmRNA isolated from total RNA using oligo dT cellulose resin. ThepBluescript II XR library construction kit (Stratagene, La Jolla,Calif.) was then used to synthesize double stranded cDNA which was thendirectionally cloned (5′NotI/3′EcoRI) into pBluescript II KS(+) vector.The T. aureum (BICC 7091) library contained approximately 1.89×10⁸clones, each with an average insert size of approximately 1300 bp.

Primers RO1210 (SEQ ID NO:40) (5′-GCT GGT TGG ACT TTG GAC ATG ATT GGATCC-3′) and RO1211 (SEQ ID NO:41) (5′-TAC ATT GGC AGG CCA ACC ATG TAGAGA ACG-3′) were designed to amplify 5′ and 3′ sequences, respectively.RO1210/RO899 (SEQ ID NO:11) and RO1211/RO898(SEQ ID NO:7) were set upwith cDNA Polymerase (Clonetech, Palo Alto, Calif.), 5 ul of cDNA fromthe library under the same conditions as described for isolating theoriginal fragment earlier in this example. After cloning and sequencingof fragments an additional internal primer RO1214 (SEQ ID NO:42) (5′-GGATTC AAT CAT GTC CAA AGT CCA ACC AGC-3′) with RO898 from the vector wasused to identify the 5′ end of the gene. In a 50 μl reaction, 10 pmol ofeach primer with 5ul of library DNA as target with Platimum Taq™ PCRx(Clonetech, Palo Alto, Calif.) with MgSO₄ was used according to themanufacturer's protocol. The cycling protocol was as follows: an initialmelt of 94° C. for 5 minutes followed by 35 cycles of 94° C. for 45seconds, 55° C. for 30 seconds, 68° C. for 2 minutes and an extensioncycle of 72° C. for 7 minutes.

The full-length Δ4-desaturase from T. aureum (BICC 7091) was isolatedwith 5′ primer RO1223 (SEQ ID NO:43) (5′-TCT GAT GAA TTC ATG ACG GCC GGATTT GAA GAA G-3′) and 3′ primer RO1224 (SEQ ID NO:44) (5′-GTC TAG CTCGAG TTA GTT CTT GTC CCA GGC AGG CA-3′) with added restriction sitesEcoRI and XhoI (underlined), respectively, added for cloning purposes.In a 50 μl reaction, 10 pmol of each primer, with 5 ul of library DNA astarget, with Platimum TAQ™ PCRx (Clonetech, Palo Alto, Calif.) withMgSO₄ according to the manufacturer's protocol, were used. The cyclingprotocol was as follows: an initial melt of 94° C. for 5 minutesfollowed by 35 cycles of 94° C. for 45 seconds, 55° C. for 30 seconds,68° C. for 2 minutes and an extension cycle of 72° C. for 7 minutes. Thesingle band was separated on an agarose gel, purified, cut with EcoRIand XhoI, and ligated to pYX242 linearized with the same enzymes.Sequence analysis of the full-length clone designated, pRTA11 (see FIG.18) (SEQ ID NO:45) revealed an open reading frame of 1542 base pairsencoding a protein of 513 amino acids (see FIG. 19) (SEQ ID NO:46).(Plasmid pRTA11 was deposited with the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110 on Mar. 27, 2002 and wasaccorded accession numbers PTA-4187.)

EXAMPLE VIII Expression of Putative Δ4-Desaturases pRSA1 and pRTA11 inYeast

Both plasmids were transformed into competent Saccharomiyces cerevisiaestrain 334 and grown as described in Example IV with either 50 μM ADA orω3-DPA. As shown in Table 4, both ω6-DPA and DHA were produced when 334(pRSA1) or (pRTA11) was grown with ADA or ω3-DPA, which are the productsof a Δ4-desaturation.

TABLE 4 Fatty Acid as a Percentage of Total Lipid Extracted from Yeast22:4(n − 6) 22:5(n − 6) 22:5(n − 3) 22:6(n − 3) Clone IncorporatedProduced Incorporated Produced PYX242 15.03 0 20.46 0.25 (control)PYX242 55.36 0 62.98 0.42 (control) PRTA11 50.73 5.42 42.39 9.17 Key:22:4(n − 6) = Adrenic acid 22:5(n − 3) = ω3-Docosapentaenoic acid 22:5(n− 6) = ω6-Docosapentaenoic acid 22:6(n − 3) = Docosahexaenoic acidWhen the percent conversion of the substrate to product was calculatedas described in Table 5, the preferred substrate, by virtue of thehigher percent conversion, was the ω3-DPA to produce DHA. This datashows clearly that these plasmids also encode Δ4-desaturases.

TABLE 5 Percent Conversion of Two Substrates to Product 50 uM 50 uMClone 22:4n − 6 22:5n − 3 PYX242 0 1.2 (control) PRSA1 3.64 9.7 PYX242 00.66 (control) PRTA11 9.65 17.78 22:4n − 6 to 22:5n − 6 (Adrenic acid toω6-Docosapentaenoic acid) 22:5n − 3 to 22:6n − 3 (ω3-Docosapentaenoicacid to Docosahexaenoic acid)

EXAMPLE IX Demonstration of Co-expression of a Δ4-Desaturase with aMouse Elongase in Yeast

As described in Example V, the T. aureum (ATCC 34304) Δ4-desaturase wasco-transformed with the mouse elongase pRMELO4 (recloned from theplasmid pRAE84 into pYES2).

Table 6 shows that when 10 μM of the substrate EPA (20:5n-3) was added,the elongase was able to add two carbons to EPA to produce ω3-DPA, andthe desaturase converted ω3-DPA to DHA. No DHA was produced by thecontrol transformation 334(pYX242/pYES2). A small amount of ω3-DPA wasseen in the control, but was a contaminant of the added substrate EPA.Thus, T. aureum Δ4-desaturase was able to produce a product in aheterologous expression system that was the product of anotherheterologous enzyme (the mouse elongase) from the PUFA biosyntheticpathway to produce the expected PUFA. This demonstrates thatΔ4-desaturase can indeed work with other heterologous enzymes in thePUFA pathway in a heterologous expression system such as yeast.

TABLE 6 Fatty Acid (μg) Extracted Lipid from Yeast DHA ω3-DPA ProducedEPA Produced by by Clone Incorporated elongase desaturase PYX242/ 59.382.54 0 PYES2 (control) PRTA7/ 47.04 14.76 1.55 PRMELO4 (mouse elongase)10 μM substrate added.

EXAMPLE IX Isolation of a Novel Desaturase Gene from the AlgaeIsochrysis galbana (CCMP1323)

The fatty acid composition of the algae Isochrysis galbana (I. galbana)(CCMP 1323) was investigated to determine the polyunsaturated fattyacids (PUFAs) produced by this organism. This algae showed a substantialamount of long chain PUFA including omega 3-docosapentaenoic acid (omega3-DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). In fact DHA waspresent in the highest amount representing 19% of the total lipid. Thus,I. galbana was predicted to possess a Δ4-desaturase capable ofconverting omega 3-DPA to DHA. The goal was therefore to isolate thepredicted Δ4-desaturase gene from I. galbana, and to verify thefunctionality of the enzyme by expression in an alternate host.

Frozen pellets of I. galbana were obtained from Provasoli-GuillardNational Center for Culture of Marine Phytoplankton (CCMP, West BoothbayHarbor, Me.). These pellets were crushed in liquid nitrogen and totalRNA was extracted from I. galbana by using the QIAGEN RNEASY MAXI Kit(Qiagen, as per manufacturers instructions. From this total RNA, mRNAwas isolated using oligo dT cellulose resin, which was then used for theconstruction of a cDNA library using the pBluescript II XR libraryconstruction kit (Stratagene, La Jolla, Calif.). The cDNA thus producedwas directionally cloned (5′NotI/3′EcoRI) into pBluescript II KS (+)vector. The I. galbana library contained approximately 9.4×10⁴ clonesper μl, each with an average insert size of approximately 1300 bp. Twothousand primary clones from this library were sequenced from the 5′ endusing the M13 forward primer (SEQ NO ID:47) (5′-AGC GGA TAA CAA TTT CACACA GG-3′). Sequencing was carried out using the ABI BIGDYE sequencingkit (Applied Biosystems, CA) and the MEGABASE Capillary DNA sequencer(Amersham biosciences, Piscataway, N.J.).

A 647 bp clone containing the 3′ end of this novel Δ4-desaturasedesignated ‘iso25-Δ09’ was obtained from sequencing of the 2000 libraryclones. This fragment shared ˜30% amino acid sequence identity withother known delta 5 and delta 6 desaturases. Since this fragment did notcontain the stop codon of the gene, additional clones containing the 3′end of this gene were obtained by PCR amplification of the cDNA library(template) using the 3′-end vector primer RO899 (SEQ ID NO:11) andRO1270 (SEQ ID NO:48) (5′-CAC CTG GCT CGA GTC GAC GAT GAT GG-3′). PCRamplification was carried out using Platinum Taq (HF) DNA polymerase(Invitrogen, Carlsbad, Calif.). Amplification was carried out in a 50 μltotal volume containing: 1 μl of the cDNA library ligation mixture, PCRbuffer containing 20 mM Tris-Cl, pH 8.4, 50 mM KCl (finalconcentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmoleof each primer, 1.5 mM MgSO₄, and 0.5 μl of Platinum Taq (HF) DNApolymerase. Amplification was carried out as follows using the PerkinElmer 9600 machine: initial melt at 94° C. for 2 minutes followed by 5cycles of 94° C. for 30 seconds and 72° C. for 3 minutes, 10 cycles of94° C. 30 seconds, 70° C. for 30 seconds, and 72° C. for 3 minutes and20 cycles of 94° C. for 30 seconds, 68° C. for 30 seconds and 72° C. for3 minutes, followed by an extension of 72° C. for 10 minutes. From thisamplification no bands were visible which might have been due to the lowamounts of this gene in the library. Thus 2 μl of this PCR reaction wasused as a template for a second PCR reaction involving Platinum Taq (HF)DNA polymerase under that same PCR components as described above.However, this time amplification was carried out as follows: initialdenaturation at 94° C. for 3 minute, followed by 30 cycles of thefollowing: 94° C. for 45 sec, 55° C. for 30 sec, 68° C. for 2 min. Thereaction was terminated at 4° C. A 670 bp PCR band was thus obtainedwhich was gel purified, and cloned into PCR-Blunt vector (Invitrogen,Carlsbad, Calif.). The recombinant plasmids were transformed into TOP10supercompetent cells (Invitrogen, Carlsbad, Calif.), and clones weresequenced and analyzed. Clones ‘iso25-A09-6’ and iso25-A09-1’ were thusobtained that contained the 3′ end of the gene along with the ‘TAA’ stopcodon and the poly-A tail. This clone did overlap with the original‘iso25-A09’ fragment.

To isolate the 5′ end of this gene, RACE (rapid amplification of cDNAends) ready cDNA was used as a target for the reactions. To prepare thismaterial, approximately 5 μg of total RNA was used according themanufacturer's direction with the GENRACER™ kit (Invitrogen, Carlsbad,Calif.) and SUPERSCRIPT II™ enzyme (Invitrogen, Carlsbad, Calif.) forreverse transcription to produce cDNA target. This cDNA was then used asa template for a PCR reaction involving 30 pmol GENRACER™ 5′ primer (SEQID NO:30) (5′-CGA CTG GAG CAC GAG GAC ACT GA-3′) in combination with 10pmols of any one of the following gene-specific primers:

RO1286 5′- CGT ACC CGG TGC AAT AGA AGG (SEQ ID NO: 49) TGA G -3′ RO12875′- CCA TCA TCG TCG ACT CGA GCC (SEQ ID NO: 50) AGG TG -3′ RO1288 5′-TGT GGA GCC ATG TGG TGC TCG (SEQ ID NO: 51) ATC TG -3′ RO1271 5′- TGTGGA GCC ATG TGG TGC TCG (SEQ ID NO: 52) ATC TG -3′PCR amplification was carried out using Platinum Taq DNA polymerase(Invitrogen, Carlsbad, Calif.) in a 50 μl total volume containing: 1 μlof the RACE-cDNA, PCR buffer containing 20 mM Tris-Cl, pH 8.4, 50 mM KCl(final concentration), 200 μM each deoxyribonucleotide triphosphate, 1.5mM MgSO₄, and 0.5 μl of Platinum Taq DNA polymerase. Amplification wascarried out as follows using the Perkin Elmer 9600 machine: initial meltat 94° C. for 2 minutes followed by 5 cycles of 94° C. for 30 secondsand 72° C. for 3 minutes, 10 cycles of 94° C. 30 seconds, 70° C. for 30seconds, and 72° C. for 3 minutes and 20 cycles of 94° C. for 30seconds, 68° C. for 30 seconds and 72° C. for 3 minutes, followed by anextension of 72° C. for 10 minutes. All these primer combinationsresulted in bands, which were gel purified, filled-in with T4-DNApolymerase, cloned into PCR-blunt vector and transformed into TOP 10supercompetent cells. Sequencing of these clones like ‘iso25-A09-33-5’,‘iso25-A09-31-3’, iso25-A09-30-1’ and iso25-Δ09-32-3’ revealed the 5′end of this gene containing the ‘ATG’ start site, the cytochrome b5domain and two histidine boxes. These clones overlapped each other andalso overlapped the original ‘iso25-Δ09’ fragment that contained thethird histidine box.

To isolate the full length of this gene both genomic DNA, as well ascDNA obtained (from RACE), were used as templates in PCR reactions withthe following primers:

-   RO 1400 (SEQ ID NO:52)-   5′-TCA ACA GAA TTC ATG TGC AAC GCG GCG CAG GTC GAG ACG CAG-3′    (This forward primer contained an EcoRI site (underlined) along with    the ‘ATG’ start site (bold) suitable for cloning into the yeast    expression vector pYX242).

RO 1401 5′- AAA AGA AAG CTT TTA GTC CGC (SEQ ID NO: 54) CTT GAC CGT GTCGAC CAA AGC -3′(This reverse primer contained a HindIII site (underlined) along withthe ‘TAA’ stop site (bold) for cloning into pYX242). PCR amplificationwas carried out using Advantage-GC cDNA polymerase (Clonetech, PaloAlto, Calif.) in a 50 μl total volume containing: 1 μl of the RACE-cDNAor 2 μl of genomic DNA, PCR buffer containing [40 mM Tricine-KOH pH 9.2,15 mM KOAc (final concentration), 3.5 mM Mg(OAc)₂, 5% DMSO, 3.75 μg/mlBSA, 200 μM each deoxyribonucleotide triphosphate, 1M GC-melt, and 1 μlof Advantage-GC cDNA polymerase. The thermocycling protocol included aninitial denaturation at 94° C. for 1 mm, followed by 30 cycles of thefollowing a final extension at 68° C. for 5 minutes, followed bytermination at 4° C.

A ˜1.35 kb band was obtained which was gel purified, digested with therestriction enzymes EcoRI/HindIII for 2 hours, cleaned through theQiaQuick PCR purification kit (Qiagen, Valencia, Calif.), and clonedinto the pYX242 yeast expression vector (Novagen, Madison, Wis.)previously digested with EcoRI/HindIII. This construct was labeled pRIG6and consisted of the ‘iso25-A09’ full length gene isolated fromRACE-derived cDNA and the pYX242 vector. This was transformed into yeastSC334 for expression studies.

The full length gene of ‘iso25-A09’ present in pRIG6 was 1302 bp inlength (SEQ ID NO:54) (FIG. 20) and encoded a protein of 433 amino acids(SEQ ID NO:55) (FIG. 21). A tFastA search of the deduced proteinsequence of this gene showed the protein to have 30.6% identity with theΔ5-desaturase from I. galbana (U.S. patent application Ser. No.10/054,534 incorporated in its entirety by reference). Also, thepredicted protein of this gene was 30.8% identical to the Δ4-desaturasefrom Thraustochytrium aureum (ATCC 34304)(FIG. 22). (Further, the DNAsequence of the gene was found to exhibit 42.37% sequence identity tothe nucleotide sequence encoding the T. aureum (ATCC 34304))Δ4-desaturase, 43% identity to the nucleotide sequence encoding the S.aggregatum (ATCC 28209) Δ4-desaturase, and 39.7% identity to thenucleotide sequence encoding the T. aureum (BICC7091) Δ4-desaturasesequence.) Like all front-end desaturating enzyme genes like Δ5- andΔ6-desaturase, this gene contains a cytochrome b5 domain within the5′-end of its sequence. This cytochrome b5 is thought to function as theimmediate electron donor for the desaturases, and functions in a numberof oxidation-reduction reactions involving NADH-dependent desaturation.This gene also possessed the three histidine-rich motifs that arepresent in all membrane-bound desaturases. These are present at position153 to 158 (HMGGH) (SEQ ID NO:71), 188 to 193 (HNKHH) (SEQ ID NO:72),and 347 to 352 (QIEHH) (SEQ ID NO:73). These histidine-rich boxes arebelieved to co-ordinate the diiron-oxo structure at the enzyme's activesite, and are necessary for enzyme activity. These features areconsistent with this gene product being a member of the membrane-bounddesaturase/hydroxylase family of the diiron-oxo proteins (3) and alsobeing a front-end desaturating enzyme. The G+C content of this gene is64.2%.

EXAMPLE X Expression of pRIG6, a Novel Desaturase from Isochrysisgalbana (CCMP 1323), in Yeast

To determine the substrate specificity and the class of reactioncatalyzed by a novel desaturase from I. galbana, the pRIG6 construct washeterologously expressed in a Saccharomyces cerevisiae (SC334), asdescribed below. Since S. cerevisiae cannot synthesize fatty acidsbeyond oleic acid (OA, 18:1 n-9), it is an ideal system to use todetermine enzyme activity on substrates longer than OA since nobackground enzyme activity will be detected. Here, substrates can beexogenously supplied to the host, taken up by the cell and acted on bythe expressed protein of the transformed gene.

Clone pRIG6, which consisted of the full-length ‘iso25-A09’ desaturasefrom I. galbana cloned into pYX242, was transformed into Saccharomycescerevisiae (SC334) using the Alkali-Cation Yeast Transformation kit (BIO101, Vista, Calif.). Transformants were selected for leucine auxotrophyon media lacking leucine (DOB [-Leu]). To detect the specific desaturaseactivity of these clones, transformants were grown in the presence of 50μM specific fatty acid substrates as listed below:

-   a. Linoleic acid (LA, 18:2n-6)—conversion to α-linolenic acid (ALA,    18:3n-3) indicates Δ15-desaturase activity; conversion to    gamma-linolenic acid indicates Δ6-desaturase activity.-   b. Dihomo-gamma-linolenic acid (20:3n-6)—conversion to    eicosatetraenoic acid (ETA, 20:4n-3) indicates Δ17-desaturase    activity; conversion to arachidonic acid (ARA, 20:4n-6) indicates    Δ5-desaturase activity.-   c. Omega-6-eicosadienoic acid (20:2n-6)—conversion to    Dihomo-gamma-linolenic acid (20:3n-6) indicates Δ8-desaturase    activity.-   d. Adrenic acid (22:4n-6)—conversion to ω6-docosapentaenoic acid    (22:5n-6) indicates Δ4-desaturase activity.-   e. Omega 3-docosapentaenoic acid (22:5n-3)—conversion to    Docosahexaenoic acid (22:6n-3) indicates Δ4-desaturase activity.    The negative control strain consisted of S. cerevisiae transformed    with the pYX242 vector, and these cultures were grown simultaneously    and analyzed.

The cultures were vigorously agitated (250 rpm) and grown for 48 hours a24° C. in the presence of 50 μM (final concentration) of the varioussubstrates (Table 7). The cells were spun down, washed once in distilledwater, and the pellets vortexed in methanol; chloroform was added alongwith tridecanoin (as an internal standard). These mixtures wereincubated for at least an hour at room temperature, or at 4° C.overnight. The chloroform layer was extracted and filtered through aWhatman filter with 1 gm anhydrous sodium sulfate to remove particulatesand residual water. The organic solvents were evaporated at 40° C. undera stream of nitrogen. The extracted lipids were then derivitized tofatty acid methyl esters (FAME) for gas chromatography analysis (GC) byadding 2 ml 0.5 N potassium hydroxide in methanol to a closed tube. Thesamples were heated to 95° C.–100° C. for 30 minutes and cooled to roomtemperature. Approximately 2 ml 14% borontrifluoride in methanol wasadded and the heating repeated. After the extracted lipid mixturecooled, 2 ml of water and 1 ml of hexane were added to extract the FAMEfor analysis by GC. The percent conversion was calculated using theformula:

${\%\mspace{14mu}{Conversion}} = {\frac{\lbrack {\%\mspace{14mu}{Product}} \rbrack}{\lbrack {{\%\mspace{14mu}{Product}} + {\%\mspace{14mu}{Substrate}}} \rbrack} \times 100}$

Table 7 shows the substrate specificity of the novel desaturaseexpressed in yeast. Here, the expressed pRIG6 clone was capable ofconverting 15.3% of ω3-docosapentaenoic acid (22:5n-3) todocosahexaenoic acid (22:5 n-3), indicating that the gene was aΔ4-desaturase. In addition, this enzyme was capable of converting 11% ofadrenic acid (22:4n-6) to ω6-docosapentaenoic acid (22:5n-6), which alsoindicated Δ4-desaturase activity.

The fatty acids of interest are represented as a percentage of the totallipids extracted from yeast. GC/MS was employed to identify theproducts. Under these conditions, the clones did not exhibit otherdesaturase activities. This confirmed the gene isolated to be a novelΔ4-desaturase gene. No background substrate conversion was detected withusing just the vector alone. This data indicates that this novelΔ4-desaturase can be expressed in a heterologous system and would thusbe useful in the production of transgenic oil containing DHA.

TABLE 7 Isochrysis galbana (CCMP 1323) Delta 4-Desaturase Expression inBaker's Yeast at 24° C. Desaturase Substrate* Substrate % ConversionClone activity Incorpor. Produced of Substrate pRIG6 Δ6 LA GLA 0 (8.35%)(0%) (pYX242 + Delta 4 Δ5 DGLA AA 0 (16.34%) (0.29%) Δ8 ω6-EDA DGLA 0(19.53%) (0%) Δ4 ADA ω6-DPA   11% (23.93%) (3.15%) Δ4 ω3-DPA DHA 15.3%(32.57%) (5.89%) Control Δ6 LA GLA 0 (9.18%) (0%) (pYX242) Δ5 DGLA AA 0(10.5%) (0%) Δ8 ω6-EDA DGLA 0 (16.56%) (0%) Δ4 ADA ω6-DPA 0 (15.55%)(0%) Δ4 ω3-DPA DHA 0 (26.03%) (0.29%) *50 μM substrate used Numbers inparenthesis represent fatty acid as a percentage of total lipids fromyeast Key: LA = Linoleic acid (18:2n − 6) GLA = Gamma-linolenic acid(18:3n − 6) DGLA = Dihomo-gamma-linolenic acid (20:3n − 6) AA =Arachidonic acid (20:4n − 6) ω6-EDA = omega-6 Eicosadienoic acid (20:2n− 6) ADA = Adrenic acid (22:4n − 6) ω3-DPA = omega-3 Docosapentaenoicacid (22:5n − 6) ω6-DPA = omega-6 Docosapentaenoic acid (22:5n − 3) DHA= Docosahexaenoic acid (22:6n − 3)Nutritional Compositions

The PUFAs described in the Detailed Description may be utilized invarious nutritional supplements, infant formulations, nutritionalsubstitutes and other nutritional solutions.

I. Infant Formulations

A. ISOMIL® Soy Formula with Iron:

Usage: As a beverage for infants, children and adults with an allergy orsensitivity to cows milk. A feeding for patients with disorders forwhich lactose should be avoided: lactase deficiency, lactose intoleranceand galactosemia.

Features:

-   -   Soy protein isolate to avoid symptoms of cow's-milk-protein        allergy or sensitivity.    -   Lactose-free formulation to avoid lactose-associated diarrhea.    -   Low osmolality (240 mOs/kg water) to reduce risk of osmotic        diarrhea.    -   Dual carbohydrates (corn syrup and sucrose) designed to enhance        carbohydrate absorption and reduce the risk of exceeding the        absorptive capacity of the damaged gut.    -   1.8 mg of Iron (as ferrous sulfate) per 100 Calories to help        prevent iron deficiency.    -   Recommended levels of vitamins and minerals.    -   Vegetable oils to provide recommended levels of essential fatty        acids.    -   Milk-white color, milk-like consistency and pleasant aroma.        Ingredients: (Pareve) 85% water, 4.9% corn syrup, 2.6% sugar        (sucrose), 2.1% soy oil, 1.9% soy protein isolate, 1.4% coconut        oil, 0.15% calcium citrate, 0.11% calcium phosphate tribasic,        potassium citrate, potassium phosphate monobasic, potassium        chloride, mono- and disglycerides, soy lecithin, carrageenan,        ascorbic acid, L-methionine, magnesium chloride, potassium        phosphate dibasic, sodium chloride, choline chloride, taurine,        ferrous sulfate, m-inositol, alpha-tocopheryl acetate, zinc        sulfate, L-carnitine, niacinamide, calcium pantothenate, cupric        sulfate, vitamin A palmitate, thiamine chloride hydrochloride,        riboflavin, pyridoxine hydrochloride, folic acid, manganese        sulfate, potassium iodide, phylloquinone, biotin, sodium        selenite, vitamin D3 and cyanocobalamin.        B. Isomil® DF Soy Formula For Diarrhea:        Usage: As a short-term feeding for the dietary management of        diarrhea in infants and toddlers.        Features:    -   First infant formula to contain added dietary fiber from soy        fiber specifically for diarrhea management.    -   Clinically shown to reduce the duration of loose, watery stools        during mild to severe diarrhea in infants.    -   Nutritionally complete to meet the nutritional needs of the        infant.    -   Soy protein isolate with added L-methionine meets or exceeds an        infant's requirement for all essential amino acids.    -   Lactose-free formulation to avoid lactose-associated diarrhea.    -   Low osmolality (240 mOsm/kg water) to reduce the risk of osmotic        diarrhea.    -   Dual carbohydrates (corn syrup and sucrose) designed to enhance        carbohydrate absorption and reduce the risk of exceeding the        absorptive capacity of the damaged gut.    -   Meets or exceeds the vitamin and mineral levels recommended by        the Committee on Nutrition of the American Academy of Pediatrics        and required by the Infant Formula Act.    -   1.8 mg of iron (as ferrous sulfate) per 100 Calories to help        prevent iron deficiency.    -   Vegetable oils to provide recommended levels of essential fatty        acids.        Ingredients: (Pareve) 86% water, 4.8% corn syrup, 2.5% sugar        (sucrose), 2.1% soy oil, 2.0% soy protein isolate, 1.4% coconut        oil, 0.77% soy fiber, 0.12% calcium citrate, 0.11% calcium        phosphate tribasic, 0.10% potassium citrate, potassium chloride,        potassium phosphate monobasic, mono and diglycerides, soy        lecithin, carrageenan, magnesium chloride, ascorbic acid,        L-methionine, potassium phosphate dibasic, sodium chloride,        choline chloride, taurine, ferrous sulfate, m-inositol,        alpha-tocopheryl acetate, zinc sulfate, L-carnitine,        niacinamide, calcium pantothenate, cupric sulfate, vitamin A        palmitate, thiamine chloride hydrochloride, riboflavin,        pyridoxine hydrochloride, folic acid, manganese sulfate,        potassium iodide, phylloquinone, biotin, sodium selenite,        vitamin D3 and cyanocobalamin.        C. ISOMIL® SF Sucrose-Free Soy Formula With Iron:        Usage: As a beverage for infants, children and adults with an        allergy or sensitivity to cow's-milk protein or an intolerance        to sucrose. A feeding for patients with disorders for which        lactose and sucrose should be avoided.        Features:    -   Soy protein isolate to avoid symptoms of cow's-milk-protein        allergy or sensitivity.    -   Lactose-free formulation to avoid lactose-associated diarrhea        (carbohydrate source is Polycose® Glucose Polymers).    -   Sucrose free for the patient who cannot tolerate sucrose.    -   Low osmolality (180 mOsm/kg water) to reduce risk of osmotic        diarrhea.    -   1.8 mg of iron (as ferrous sulfate) per 100 Calories to help        prevent iron deficiency.    -   Recommended levels of vitamins and minerals.    -   Vegetable oils to provide recommended levels of essential fatty        acids.    -   Milk-white color, milk-like consistency and pleasant aroma.        Ingredients: (Pareve) 75% water, 11.8% hydrolized cornstarch,        4.1% soy oil, 4.1% soy protein isolate, 2.8% coconut oil, 1.0%        modified cornstarch, 0.38% calcium phosphate tribasic, 0.17%        potassium citrate, 0.13% potassium chloride, mono- and        diglycerides, soy lecithin, magnesium chloride, abscorbic acid,        L-methionine, calcium carbonate, sodium chloride, choline        chloride, carrageenan, taurine, ferrous sulfate, m-inositol,        alpha-tocopheryl acetate, zinc sulfate,L-carnitine, niacinamide,        calcium pantothenate, cupric sulfate, vitamin A palmitate,        thiamine chloride hydrochloride, riboflavin, pyridoxine        hydrochloride, folic acid, manganese sulfate, potassium iodide,        phylloquinone, biotin, sodium selenite, vitamin D3 and        cyanocobalamin.        D. ISOMIL® 20 Soy Formula with Iron Ready to Feed, 20 Cal/fl        oz.:        Usage: When a soy feeding is desired.        Ingredients: (Pareve) 85% water, 4.9% corn syrup, 2.6%        sugar(sucrose), 2.1% soy oil, 1.9% soy protein isolate, 1.4%        coconut oil, 0.15% calcium citrate, 0.11% calcium phosphate        tribasic, potassium citrate, potassium phosphate monobasic,        potassium chloride, mono- and diglycerides, soy lecithin,        carrageenan, abscorbic acid, L-methionine, magnesium chloride,        potassium phosphate dibasic, sodium chloride, choline chloride,        taurine, ferrous sulfate, m-inositol, alpha-tocopheryl acetate,        zinc sulfate, L-carnitine, niacinamide, calcium pantothenate,        cupric sulfate, vitamin A palmitate, thiamine chloride        hydrochloride, riboflavin, pyridoxine hydrochloride, folic acid,        manganese sulfate, potassium iodide, phylloquinone, biotin,        sodium selenite, vitamin D3 and cyanocobalamin.        E. SIMILAC® Infant Formula:        Usage: When an infant formula is needed: if the decision is made        to discontinue breastfeeding before age 1 year, if a supplement        to breastfeeding is needed or as a routine feeding if        breastfeeding is not adopted.        Features:    -   Protein of appropriate quality and quantity for good growth;        heat-denatured, which reduces the risk of milk-associated        enteric blood loss.    -   Fat from a blend of vegetable oils (doubly homogenized),        providing essential linoleic acid that is easily absorbed.    -   Carbohydrate as lactose in proportion similar to that of human        milk.    -   Low renal solute load to minimize stress on developing organs.    -   Powder, Concentrated Liquid and Ready To Feed forms.        Ingredients: (-D) Water, nonfat milk, lactose, soy oil, coconut        oil, mono- and diglycerides, soy lecithin, abscorbic acid,        carrageenan, choline chloride, taurine, m-inositol,        alpha-tocopheryl acetate, zinc sulfate, niacinamide, ferrous        sulfate, calcium pantothenate, cupric sulfate, vitamin A        palmitate, thiamine chloride hydrochloride, riboflavin,        pyridoxine hydrochloride, folic acid, manganese sulfate,        phylloquinone, biotin, sodium selenite, vitamin D3 and        cyanocobalamin.        F. SIMILAC® NeoCare Premature Infant Formula with Iron:        Usage: For premature infants' special nutritional needs after        hospital discharge. Similac NeoCare is a nutritionally complete        formula developed to provide premature infants with extra        calories, protein, vitamins and minerals needed to promote        catch-up growth and support development.        Features:    -   Reduces the need for caloric and vitamin supplementation. More        calories (22 Cal/fl oz) than standard term formulas (20 Cal/fl        oz).    -   Highly absorbed fat blend, with medium-chain triglycerides        (MCToil) to help meet the special digestive needs of premature        infants.    -   Higher levels of protein, vitamins and minerals per 100 calories        to extend the nutritional support initiated in-hospital.    -   More calcium and phosphorus for improved bone mineralization.        Ingredients: -D Corn syrup solids, nonfat milk, lactose, whey        protein concentrate, soy oil, high-oleic safflower oil,        fractionated coconut oil (medium chain triglycerides), coconut        oil, potassium citrate, calcium phosphate tribasic, calcium        carbonate, ascorbic acid, magnesium chloride, potassium        chloride, sodium chloride, taurine, ferrous sulfate, m-inositol,        choline chloride, ascorbyl palmitate, L-carnitine,        alpha-tocopheryl acetate, zinc sulfate, niacinamide, mixed        tocopherols, sodium citrate, calcium pantothenate, cupric        sulfate, thiamine chloride hydrochloride, vitamin A palmitate,        beta carotene, riboflavin, pyridoxine hydrochloride, folic acid,        manganese sulfate, phylloquinone, biotin, sodium selenite,        vitamin D3 and cyanocobalamin.        G. SIMILAC Natural Care Low-Iron Human Milk Fortifier Ready To        Use, 24 Cal/fl oz.:        Usage: Designed to be mixed with human milk or to be fed        alternatively with human milk to low-birth-weight infants.        Ingredients: -D Water, nonfat milk, hydrolyzed cornstarch,        lactose, fractionated coconut oil (medium-chain triglycerides),        whey protein concentrate, soy oil, coconut oil, calcium        phosphate tribasic, potassium citrate, magnesium chloride,        sodium citrate, ascorbic acid, calcium carbonate, mono and        diglycerides, soy lecithin, carrageenan, choline chloride,        m-inositol, taurine, niacinamide, L-carnitine, alpha tocopheryl        acetate, zinc sulfate, potassium chloride, calcium pantothenate,        ferrous sulfate, cupric sulfate, riboflavin, vitamin A        palmitate, thiamine chloride hydrochloride, pyridoxine        hydrochloride, biotin, folic acid, manganese sulfate,        phylloquinone, vitamin D3, sodium selenite and cyanocobalamin.

Various PUFAs of this invention can be substituted and/or added to theinfant formulae described above and to other infant formulae known tothose in the art.

II. Nutritional Formulations

A. ENSURE®

Usage: ENSURE is a low-residue liquid food designed primarily as an oralnutritional supplement to be used with or between meals or, inappropriate amounts, as a meal replacement. ENSURE is lactose- andgluten-free, and is suitable for use in modified diets, includinglow-cholesterol diets. Although it is primarily an oral supplement, itcan be fed by tube.Patient Conditions:

-   -   For patients on modified diets    -   For elderly patients at nutrition risk    -   For patients with involuntary weight loss    -   For patients recovering from illness or surgery    -   For patients who need a low-residue diet        Ingredients: -D Water, Sugar (Sucrose), Maltodextrin (Corn),        Calcium and Sodium Caseinates, High-Oleic Safflower Oil, Soy        Protein Isolate, Soy Oil, Canola Oil, Potassium Citrate, Calcium        Phosphate Tribasic, Sodium Citrate, Magnesium Chloride,        Magnesium Phosphate Dibasic, Artificial Flavor, Sodium Chloride,        Soy Lecithin, Choline Chloride, Ascorbic Acid, Carrageenan, Zinc        Sulfate, Ferrous Sulfate, Alpha-Tocopheryl Acetate, Gellan Gum,        Niacinamide, Calcium Pantothenate, Manganese Sulfate, Cupric        Sulfate, Vitamin A Palmitate, Thiamine Chloride Hydrochloride,        Pyridoxine Hydrochloride, Riboflavin, Folic Acid, Sodium        Molybdate, Chromium Chloride, Biotin, Potassium Iodide, Sodium        Selenate.        B. ENSURE® BARS:        Usage: ENSURE BARS are complete, balanced nutrition for        supplemental use between or with meals. They provide a        delicious, nutrient-rich alternative to other snacks. ENSURE        BARS contain <1 g lactose/bar, and Chocolate Fudge Brownie        flavor is gluten-free. (Honey Graham Crunch flavor contains        gluten.)        Patient Conditions:    -   For patients who need extra calories, protein, vitamins and        minerals.    -   Especially useful for people who do not take in enough calories        and nutrients.    -   For people who have the ability to chew and swallow    -   Not to be used by anyone with a peanut allergy or any type of        allergy to nuts.        Ingredients: Honey Graham Crunch—High-Fructose Corn Syrup, Soy        Protein Isolate, Brown Sugar, Honey, Maltodextrin (Corn), Crisp        Rice (Milled Rice, Sugar [Sucrose], Salt [Sodium Chloride] and        Malt), Oat Bran, Partially Hydrogenated Cottonseed and Soy Oils,        Soy Polysaccharide, Glycerine, Whey Protein Concentrate,        Polydextrose, Fructose, Calcium Caseinate, Cocoa Powder,        Artificial Flavors, Canola Oil, High-Oleic Safflower Oil, Nonfat        Dry Milk, Whey Powder, Soy Lecithin and Corn Oil. Manufactured        in a facility that processes nuts.        Vitamins and Minerals: Calcium Phosphate Tribasic, Potassium        Phosphate Dibasic, Magnesium Oxide, Salt (Sodium Chloride),        Potassium Chloride, Ascorbic Acid, Ferric Orthophosphate,        Alpha-Tocopheryl Acetate, Niacinamide, Zinc Oxide, Calcium        Pantothenate, Copper Gluconate, Manganese Sulfate, Riboflavin,        Beta Carotene, Pyridoxine Hydrochloride, Thiamine Mononitrate,        Folic Acid, Biotin, Chromium Chloride, Potassium Iodide, Sodium        Selenate, Sodium Molybdate, Phylloquinone, Vitamin D3 and        Cyanocobalamin.        Protein: Honey Graham Crunch—The protein source is a blend of        soy protein isolate and milk proteins.

Soy protein isolate 74% Milk proteins 26%Fat: Honey Graham Crunch—The fat source is a blend of partiallyhydrogenated cottonseed and soybean, canola, high oleic safflower, oils,and soy lecithin.

Partially hydrogenated 76% cottonseed and soybean oil Canola oil 8%High-oleic safflower oil 8% Corn oil 4% Soy lecithin 4%Carbohydrate: Honey Graham Crunch—The carbohydrate source is acombination of high-fructose corn syrup, brown sugar, maltodextrin,honey, crisp rice, glycerine, soy polysaccharide, and oat bran.

High-fructose corn syrup 24% Brown sugar 21% Maltodextrin 12% Honey 11%Crisp rice 9% Glycerine 9% Soy Polysaccharide 7% Oat bran 7%C. ENSURE® HIGH PROTEIN:Usage: ENSURE HIGH PROTEIN is a concentrated, high-protein liquid fooddesigned for people who require additional calories, protein, vitamins,and minerals in their diets. It can be used as an oral nutritionalsupplement with or between meals or, in appropriate amounts, as a mealreplacement. ENSURE HIGH PROTEIN is lactose- and gluten-free, and issuitable for use by people recovering from general surgery or hipfractures and by patients at risk for pressure ulcers.Patient Conditions:

-   -   For patients who require additional calories, protein, vitamins,        and minerals, such as patients recovering from general surgery        or hip fractures, patients at risk for pressure ulcers, and        patients on low-cholesterol diets.        Features:    -   Low in saturated fat    -   Contains 6 g of total fat and <5 mg of cholesterol per serving    -   Rich, creamy taste    -   Excellent source of protein, calcium, and other essential        vitamins and minerals    -   For low-cholesterol diets    -   Lactose-free, easily digested        Ingredients:        Vanilla Supreme: -D Water, Sugar (Sucrose), Maltodextrin (Corn),        Calcium and Sodium Caseinates, High-Oleic Safflower Oil, Soy        Protein Isolate, Soy Oil, Canola Oil, Potassium Citrate, Calcium        Phosphate Tribasic, Sodium Citrate, Magnesium Chloride,        Magnesium Phosphate Dibasic, Artificial Flavor, Sodium Chloride,        Soy Lecithin, Choline Chloride, Ascorbic Acid, Carrageenan, Zinc        Sulfate, Ferrous Suffate, Alpha-Tocopheryl Acetate, Gellan Gum,        Niacinamide, Calcium Pantothenate, Manganese Sulfate, Cupric        Sulfate, Vitamin A Palmitate, Thiamine Chloride Hydrochloride,        Pyridoxine Hydrochloride, Riboflavin, Folic Acid, Sodium        Molybdate, Chromium Chloride, Biotin, Potassium Iodide, Sodium        Selenate, Phylloquinone, Vitamin D3 and Cyanocobalamin.        Protein:

The protein source is a blend of two high-biologic-value proteins:casein and soy.

Sodium and calcium caseinates 85% Soy protein isolate 15%Fat:

The fat source is a blend of three oils: high-oleic safflower, canola,and soy.

High-oleic safflower oil 40% Canola oil 30% Soy oil 30%The level of fat in ENSURE HIGH PROTEIN meets American Heart Association(AHA) guidelines. The 6 grams of fat in ENSURE HIGH PROTEIN represent24% of the total calories, with 2.6% of the fat being from saturatedfatty acids and 7.9% from polyunsaturated fatty acids. These values arewithin the AHA guidelines of <30% of total calories from fat, <10% ofthe calories from saturated fatty acids, and <10% of total calories frompolyunsaturated fatty acids.Carbohydrate:

ENSURE HIGH PROTEIN contains a combination of maltodextrin and sucrose.The mild sweetness and flavor variety (vanilla supreme, chocolate royal,wild berry, and banana), plus VARI-FLAVORS® Flavor Pacs in pecan,cherry, strawberry, lemon, and orange, help to prevent flavor fatigueand aid in patient compliance.

Vanilla and other nonchocolate flavors: Sucrose 60% Maltodextrin 40%Chocolate: Sucrose 70% Maltodextrin 30%D. ENSURE® LIGHTUsage: ENSURE LIGHT is a low-fat liquid food designed for use as an oralnutritional supplement with or between meals. ENSURE LIGHT is lactose-and gluten-free, and is suitable for use in modified diets, includinglow-cholesterol diets.Patient Conditions:

-   -   For normal-weight or overweight patients who need extra        nutrition in a supplement that contains 50% less fat and 20%        fewer calories than ENSURE.    -   For healthy adults who do not eat right and need extra        nutrition.        Features:    -   Low in fat and saturated fat    -   Contains 3 g of total fat per serving and <5 mg cholesterol    -   Rich, creamy taste    -   Excellent source of calcium and other essential vitamins and        minerals    -   For low-cholesterol diets    -   Lactose-free, easily digested        Ingredients:        French Vanilla: -D Water, Maltodextrin (Corn), Sugar (Sucrose),        Calcium Caseinate, High-Oleic Safflower Oil, Canola Oil,        Magnesium Chloride, Sodium Citrate, Potassium Citrate, Potassium        Phosphate Dibasic, Magnesium Phosphate Dibasic, Natural and        Artificial Flavor, Calcium Phosphate Tribasic, Cellulose Gel,        Choline Chloride, Soy Lecithin, Carrageenan, Salt (Sodium        Chloride), Ascorbic Acid, Cellulose Gum, Ferrous Sulfate,        Alpha-Tocopheryl Acetate, Zinc Sulfate, Niacinamide, Manganese        Sulfate, Calcium Pantothenate, Cupric Sulfate, Thiamine Chloride        Hydrochloride, Vitamin A Palmitate, Pyridoxine Hydrochloride,        Riboflavin, Chromium Chloride, Folic Acid, Sodium Molybdate,        Biotin, Potassium Iodide, Sodium Selenate, Phylloquinone,        Vitamin D3 and Cyanocobalamin.        Protein:        The protein source is calcium caseinate.

Calcium caseinate 100%Fat:The fat source is a blend of two oils: high-oleic safflower and canola.

High-oleic safflower oil 70% Canola oil 30%The level of fat in ENSURE LIGHT meets American Heart Association (AHA)guidelines. The 3 grams of fat in ENSURE LIGHT represent 13.5% of thetotal calories, with 1.4% of the fat being from saturated fatty acidsand. 2.6% from polyunsaturated fatty acids. These values are within theAHA guidelines of <30% of total calories from fat, <10% of the, caloriesfrom saturated fatty acids, and <10% of total calories frompolyunsaturated fatty acids.Carbohydrate:ENSURE LIGHT contains a combination of maltodextrin and sucrose. Thechocolate flavor contains corn syrup as well. The mild sweetness andflavor variety (French vanilla, chocolate supreme, strawberry swirl),plus VARI-FLAVORS® Flavor Pacs in pecan, cherry, strawberry, lemon, andorange, help to prevent flavor fatigue and aid in patient compliance.Vanilla and Other Nonchocolate Flavors:

Sucrose 51% Maltodextrin 49%Chocolate:

Sucrose 47.0% Corn Syrup 26.5% Maltodextrin 26.5%Vitamins and Minerals:An 8-fl-oz serving of ENSURE LIGHT provides at least 25% of the RDIs for24 key vitamins and minerals.Caffeine:

Chocolate flavor contains 2.1 mg caffeine/8 fl oz.

E. ENSURE PLUS®

Usage: ENSURE PLUS is a high-calorie, low-residue liquid food for usewhen extra calories and nutrients, but a normal concentration ofprotein, are needed. It is designed primarily as an oral nutritionalsupplement to be used with or between meals or, in appropriate amounts,as a meal replacement. ENSURE PLUS is lactose- and gluten-free. Althoughit is primarily an oral nutritional supplement, it can be fed by tube.Patient Conditions:

-   -   For patients who require extra calories and nutrients, but a        normal concentration of protein, in a limited volume.    -   For patients who need to gain or maintain healthy weight.        Features:    -   Rich, creamy taste    -   Good source of essential vitamins and minerals        Ingredients:        Vanilla: -D Water, Corn Syrup, Maltodextrin (Corn), Corn Oil,        Sodium and Calcium Caseinates, Sugar (Sucrose), Soy Protein        Isolate, Magnesium Chloride, Potassium Citrate, Calcium        Phosphate Tribasic, Soy Lecithin, Natural and Artificial Flavor,        Sodium Citrate, Potassium Chloride, Choline Chloride, Ascorbic        Acid, Carrageenan, Zinc Sulfate, Ferrous Sulfate,        Alpha-Tocopheryl Acetate, Niacinamide, Calcium Pantothenate,        Manganese Sulfate, Cupric Sulfate, Thiamine Chloride        Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, Vitamin A        Palmitate, Folic Acid, Biotin, Chromium Chloride, Sodium        Molybdate, Potassium Iodide, Sodium Selenite, Phylloquinone,        Cyanocobalamin and Vitamin D3.        Protein:

The protein source is a blend of two high-biologic-value proteins:casein and soy.

Sodium and calcium caseinates 84% Soy protein isolate 16%Fat:

The fat source is corn oil.

Corn oil 100%Carbohydrate:

ENSURE PLUS contains a combination of maltodextrin and sucrose. The mildsweetness and flavor variety (vanilla, chocolate, strawberry, coffee,buffer pecan, and eggnog), plus VARI-FLAVORS® Flavor Pacs in pecan,cherry, strawberry, lemon, and orange, help to prevent flavor fatigueand aid in patient compliance.

Vanilla, Strawberry, Butter Pecan, and Coffee Flavors:

Corn Syrup

-   -   39%        Maltodextrin    -   38%        Sucrose    -   23%        Chocolate and Eggnog Flavors:        Corn Syrup        36%        Maltodextrin    -   34%        Sucrose        30%        Vitamins and Minerals:

An 8-fl-oz serving of ENSURE PLUS provides at least 15% of the RDIs for25 key Vitamins and minerals.

Caffeine:

Chocolate flavor contains 3.1 mg Caffeine/8 fl oz. Coffee flavorcontains a trace amount of caffeine.

F. ENSURE PLUS® HN

Usage: ENSURE PLUS HN is a nutritionally complete high-calorie,high-nitrogen liquid food designed for people with higher calorie andprotein needs or limited volume tolerance. It may be used for oralsupplementation or for total nutritional support by tube. ENSURE PLUS HNis lactose- and gluten-free.Patient Conditions:

-   -   For patients with increased calorie and protein needs, such as        following surgery or injury.    -   For patients with limited volume tolerance and early satiety.        Features:    -   For supplemental or total nutrition    -   For oral or tube feeding    -   1.5 CaVmL,    -   High nitrogen    -   Calorically dense        Ingredients:        Vanilla: -D Water, Maltodextrin (Corn), Sodium and Calcium        Caseinates, Corn Oil, Sugar (Sucrose), Soy Protein Isolate,        Magnesium Chloride, Potassium Citrate, Calcium Phosphate        Tribasic, Soy Lecithin, Natural and Artificial Flavor, Sodium        Citrate, Choline Chloride, Ascorbic Acid, Taurine, L-Carnitine,        Zinc Sulfate, Ferrous Sulfate, Alpha-Tocopheryl Acetate,        Niacinamide, Carrageenan, Calcium Pantothenate, Manganese        Sulfate, Cupric Sulfate, Thiamine Chloride Hydrochloride,        Pyridoxine Hydrochloride, Riboflavin, Vitamin A Palmitate, Folic        Acid, Biotin, Chromium Chloride, Sodium Molybdate, Potassium        Iodide, Sodium Selenite, Phylloquinone, Cyanocobalamin and        Vitamin D3.        G. ENSURE® POWDER:        Usage: ENSURE POWDER (reconstituted with water) is a low-residu        liquid food designed primarily as an oral nutritional supplement        to be used with or between meals. ENSURE POWDER is lactose- and        gluten-free, and is suitable for use in modified diets,        including low-cholesterol diets.        Patient Conditions:    -   For patients on modified diets    -   For elderly patients at nutrition risk    -   For patients recovering from illness/surgery    -   For patients who need a low-residue diet        Features:    -   Convenient, easy to mix    -   Low in saturated fat    -   Contains 9 g of total fat and <5 mg of cholesterol per serving    -   High in vitamins and minerals    -   For low-cholesterol diets    -   Lactose-free, easily digested        Ingredients: -D Corn Syrup, Maltodextrin (Corn), Sugar        (Sucrose), Corn Oil, Sodium and Calcium Caseinates, Soy Protein        Isolate, Artificial Flavor, Potassium Citrate, Magnesium        Chloride, Sodium Citrate, Calcium Phosphate Tribasic, Potassium        Chloride, Soy Lecithin, Ascorbic Acid, Choline Chloride, Zinc        Sulfate, Ferrous Sulfate, Alpha-Tocopheryl Acetate, Niacinamide,        Calcium Pantothenate, Manganese Sulfate, Thiamine Chloride        Hydrochloride, Cupric Sulfate, Pyridoxine Hydrochloride,        Riboflavin, Vitamin A Palmitate, Folic Acid, Biotin, Sodium        Molybdate, Chromium Chloride, Potassium Iodide, Sodium Selenate,        Phylloquinone, Vitamin D3 and Cyanocobalamin.        Protein:        The protein source is a blend of two high-biologic-value        proteins: casein and soy.

Sodium and calcium caseinates 84% Soy protein isolate 16%Fat:

The fat source is corn oil.

Corn oil 100%Carbohydrate:

ENSURE POWDER contains a combination of corn syrup, maltodextrin, andsucrose. The mild sweetness of ENSURE POWDER, plus VARI-FLAVORS® FlavorPacs in pecan, cherry, strawberry, lemon, and orange, helps to preventflavor fatigue and aid in patient compliance.

Vanilla:

Corn Syrup 35% Maltodextrin 35% Sucrose 30%H. ENSURE® PUDDINGUsage: ENSURE PUDDING is a nutrient-dense supplement providing balancednutrition in a nonliquid form to be used with or between meals. It isappropriate for consistency-modified diets (e.g., soft, pureed, or fullliquid) or for people with swallowing impairments. ENSURE PUDDING isgluten-free.Patient Conditions:

-   -   For patients on consistency-modified diets (e.g., soft, pureed,        or full liquid)    -   For patients with swallowing impairments        Features:    -   Rich and creamy, good taste    -   Good source of essential vitamins and minerals    -   Convenient-needs no refrigeration    -   Gluten-free        Nutrient Profile per 5 oz: Calories 250, Protein 10.9%, Total        Fat 34.9%, Carbohydrate 54.2%        Ingredients:        Vanilla: -D Nonfat Milk, Water, Sugar (Sucrose), Partially        Hydrogenated Soybean Oil, Modified Food Starch, Magnesium        Sulfate, Sodium Stearoyl Lactylate, Sodium Phosphate Dibasic,        Artificial Flavor, Ascorbic Acid, Zinc Sulfate, Ferrous Sulfate,        Alpha-Tocopheryl Acetate, Choline Chloride, Niacinamide,        Manganese Sulfate, Calcium Pantothenate, FD&C Yellow #5,        Potassium Citrate, Cupric Sulfate, Vitamin A Palmitate, Thiamine        Chloride Hydrochloride, Pyridoxine Hydrochloride, Riboflavin,        FD&C Yellow #6, Folic Acid, Biotin, Phylloquinone, Vitamin D3        and Cyanocobalamin.        Protein:

The protein source is nonfat milk.

Nonfat milk 100%Fat:

The fat source is hydrogenated soybean oil.

Hydrogenated soybean oil 100%Carbohydrate:

ENSURE PUDDING contains a combination of sucrose and modified foodstarch. The mild sweetness and flavor variety (vanilla, chocolate,butterscotch, and tapioca) help prevent flavor fatigue. The productcontains 9.2 grams of lactose per serving.

Vanilla and Other Nonchocolate Flavors:

Sucrose

-   -   56%        Lactose    -   27%        Modified food starch    -   17%        Chocolate:        Sucrose    -   58%        Lactose    -   26%        Modified food starch    -   16%        I. ENSURE® WITH FIBER:        Usage: ENSURE WITH FIBER is a fiber-containing, nutritionally        complete liquid food designed for people who can benefit from        increased dietary fiber and nutrients. ENSURE WITH FIBER is        suitable for people who do not require a low-residue diet. It        can be fed orally or by tube, and can be used as a nutritional        supplement to a regular diet or, in appropriate amounts, as a        meal replacement. ENSURE WITH FIBER is lactose- and gluten-free,        and is suitable for use in modified diets, including        low-cholesterol diets.        Patient Conditions:    -   For patients who can benefit from increased dietary fiber and        nutrients        Features:    -   New advanced formula-low in saturated fat, higher in vitamins        and minerals    -   Contains 6 g of total fat and <5 mg of cholesterol per serving    -   Rich, creamy taste    -   Good source of fiber    -   Excellent source of essential vitamins and minerals    -   For low-cholesterol diets    -   Lactose- and gluten-free        Ingredients:        Vanilla: -D Water; Maltodextrin (Corn), Sugar (Sucrose), Sodium        and Calcium Caseinates, Oat Fiber, High-Oleic Safflower Oil,        Canola Oil, Soy Protein Isolate, Corn Oil, Soy Fiber, Calcium        Phosphate Tribasic, Magnesium Chloride, Potassium Citrate,        Cellulose Gel, Soy Lecithin, Potassium Phosphate Dibasic, Sodium        Citrate, Natural and Artificial Flavors, Choline Chloride,        Magnesium Phosphate, Ascorbic Acid, Cellulose Gum, Potassium        Chloride, Carrageenan, Ferrous Sulfate, Alpha-Tocopheryl        Acetate, Zinc Sulfate, Niacinamide, Manganese Sulfate, Calcium        Pantothenate, Cupric Sulfate, Vitamin A Palmitate, Thiamine        Chloride Hydrochloride, Pyridoxine Hydrochloride, Riboflavin,        Folic Acid, Chromium Chloride, Biotin, Sodium Molybdate,        Potassium Iodide, Sodium Selenate, Phylloquinone, Vitamin D3 and        Cyanocobalamin.        Protein:

The protein source is a blend of two high-biologic-value proteins-caseinand soy.

Sodium and calcium caseinates 80% Soy protein isolate 20%Fat:

The fat source is a blend of three oils: high-oleic safflower, canola,and corn.

High-oleic safflower oil 40% Canola oil 40% Corn oil 20%The level of fat in ENSURE WITH FIBER meets American Heart Association(AHA) guidelines. The 6 grams of fat in ENSURE WITH FIBER represent 22%of the total calories, with 2.01% of the fat being from saturated fattyacids and 6.7% from polyunsaturated fatty acids. These values are withinthe AHA guidelines of ≦30% of total calories from fat, <10% of thecalories from saturated fatty acids, and ≦10% of total calories frompolyunsaturated fatty acids.Carbohydrate:

ENSURE WITH FIBER contains a combination of maltodextrin and sucrose.The mild sweetness and flavor variety (vanilla, chocolate, and butterpecan), plus VARI-FLAVORS® Flavor Pacs in pecan, cherry, strawberry,lemon, and orange, help to prevent flavor fatigue and aid in patientcompliance.

Vanilla and Other Nonchocolate Flavors:

Maltodextrin 66% Sucrose 25% Oat Fiber 7% Soy Fiber 2%Chocolate:

Maltodextrin 55% Sucrose 36% Oat Fiber 7% Soy Fiber 2%Fiber:

The fiber blend used in ENSURE WITH FIBER consists of oat fiber and soypolysaccharide. This blend results in approximately 4 grams of totaldietary fiber per 8-fl. oz can. The ratio of insoluble to soluble fiberis 95:5.

The various nutritional supplements described above and known to othersof skill in the art can be substituted and/or supplemented with thePUFAs produced in accordance with the present invention.

J. OXEPA™ Nutritional Product

Oxepa is a low-carbohydrate, calorically dense, enteral nutritionalproduct designed for the dietary management of patients with or at riskfor ARDS. It has a unique combination of ingredients, including apatented oil blend containing eicosapentaenoic acid (EPA from fish oil),γ-linolenic acid (GLA from borage oil), and elevated antioxidant levels.

Caloric Distribution:

Caloric density is high at 1.5 Cal/mL (355 Cal/8 fl oz), to minimize thevolume required to meet energy needs.

The distribution of Calories in Oxepa is shown in Table A.

TABLE A Caloric Distribution of Oxepa per 8 fl oz. per liter % of CalCalories 355 1,500 — Fat (g) 22.2 93.7 55.2 Carbohydrate (g) 25 105.528.1 Protein (g) 14.8 62.5 16.7 Water (g) 186 785 —Fat:

-   -   Oxepa contains 22.2 g of fat per 8-fl oz serving (93.7 g/L).    -   The fat source is an oil blend of 31.8% canola oil, 25%        medium-chain triglycerides (MCTs), 20% borage oil, 20% fish oil,        and 3.2% soy lecithin. The typical fatty acid profile of Oxepa        is shown in Table B.

OXEPA™ provides a balanced amount of polyunsaturated, monounsaturated,and saturated fatty acids, as shown in Table VI.

-   -   Medium-chain trigylcerides (MCTs)—25% of the fat blend—aid        gastric emptying because they are absorbed by the intestinal        tract without emulsification by bile acids.        The various fatty acid components of Oxepa™ nutritional product        can be substituted and/or supplemented with the PUFAs produced        in accordance with this invention.

TABLE B Typical Fatty Acid Profile g/8 fl Fatty Acids % Total oz* 9/L*Caproic (6:0) 0.2 0.04 0.18 Caprylic (8:0) 14.69 3.1 13.07 Capric (10:0)11.06 2.33 9.87 Palmitic (16:0) 5.59 1.18 4.98 Palmitoleic 1.82 0.381.62 Stearic 1.94 0.39 1.64 Oleic 24.44 5.16 21.75 Linoleic 16.28 3.4414.49 α-Linolenic 3.47 0.73 3.09 γ-Linolenic 4.82 1.02 4.29Eicosapentaenoic 5.11 1.08 4.55 n-3-Docosapentaenoic 0.55 0.12 0.49Docosahexaenoic 2.27 0.48 2.02 Others 7.55 1.52 6.72Fatty acids equal approximately 95% of total fat.

TABLE C Fat Profile of Oxepa. % of total calories from fat 55.2Polyunsaturated fatty acids 31.44 g/L Monounsaturated fatty acids 25.53g/L Saturated fatty acids 32.38 g/L n − 6 to n − 3 ratio 1.75:1Cholesterol  9.49 mg/8 fl oz  40.1 mg/LCarbohydrate:

-   -   The carbohydrate content is 25.0 g per 8-fl-oz serving (105.5        g/L).    -   The carbohydrate sources are 45% maltodextrin (a complex        carbohydrate) and 55% sucrose (a simple sugar), both of which        are readily digested and absorbed.    -   The high-fat and low-carbohydrate content of Oxepa is designed        to minimize carbon dioxide (C02) production. High C02 levels can        complicate weaning in ventilator-dependent patients. The low        level of carbohydrate also may be useful for those patients who        have developed stress-induced hyperglycemia.

OXEPA™ is lactose-free.

Dietary carbohydrate, the amino acids from protein, and the glycerolmoiety of fats can be converted to glucose within the body. Throughoutthis process, the carbohydrate requirements of glucose-dependent tissues(such as the central nervous system and red blood cells) are met.However, a diet free of carbohydrates can lead to ketosis, excessivecatabolism of tissue protein, and loss of fluid and electrolytes. Theseeffects can be prevented by daily ingestion of 50 to 100 g of digestiblecarbohydrate, if caloric intake is adequate. The carbohydrate level inOxepa is also sufficient to minimize gluconeogenesis, if energy needsare being met.

Protein:

-   -   Oxepa contains 14.8 g of protein per 8-fl-oz serving (62.5 g/L).    -   The total calorie/nitrogen ratio (150:1) meets the need of        stressed patients.    -   Oxepa provides enough protein to promote anabolism and the        maintenance of lean body mass without precipitating respiratory        problems. High protein intakes are a concern in patients with        respiratory insufficiency. Although protein has little effect on        CO₂ production, a high protein diet will increase ventilatory        drive.    -   The protein sources of Oxepa are 86.8% sodium caseinate and        13.2% calcium caseinate.

The amino acid profile of the protein system in Oxepa meets or surpassesthe standard for high quality protein set by the National Academy ofSciences.

OXEPA™ is gluten-free.

1. An isolated nucleotide sequence comprising or complementary to anucleotide sequence encoding a polypeptide having Δ4-desaturaseactivity, wherein the amino acid sequence of said polypeptide has atleast 90% identity to the amino acid sequence of SEQ ID NO: 55, or afragment thereof encoding Δ4-desaturase activity.
 2. An isolatednucleotide sequence comprising or complementary to a nucleotide sequencehaving at least 90% identity to the nucleotide sequence of SEQ ID NO: 54encoding Δ4-desaturase activity, or a fragment thereof encodingΔ4-desaturase activity.
 3. The nucleotide sequence of claim 2 whereinsaid SEQ ID NO: 54 is derived from the algae Isochrysis galbana.
 4. Amethod of producing a Δ4-desaturase comprising the steps of: a)isolating a nucleotide sequence comprising or complementary to anucleotide sequence: i) encoding a polypeptide comprising the amino acidsequence having at least 90% identity to an amino acid sequence of SEQID NO:55 having Δ4-desaturase activity or ii) having at least 90%identity to the nucleotide sequence of SEQ ID NO: 54 encodingΔ4-desaturase activity; b) constructing a vector comprising: i) saidisolated nucleotide sequence operably linked to ii) a promoter; and c)introducing said vector into a host cell for a time and under conditionssufficient for expression of said desaturase, thereby producingΔ4-desaturase.
 5. A vector comprising: a) an isolated nucleotidesequence comprising or complementary to a nucleotide sequence: i)encoding a polypeptide comprising the amino acid sequence having atleast 90% identity to an amino acid sequence of SEQ ID NO: 55 havingΔ4-desaturase activity or ii) having at least 90% identity to thenucleotide sequence of SEQ ID NO: 54 encoding Δ4-desaturase activity,operably linked to b) a promoter.
 6. An isolated host cell comprisingsaid vector of claim
 5. 7. The isolated nucleotide sequence of claim 1or claim 2 wherein said nucleotide sequence encodes a Δ4-desaturasewhich utilizes a monounsaturated or polyunsaturated fatty acid as asubstrate.
 8. A method for producing a polyunsaturated fatty acidcomprising the steps of: a) isolating said nucleotide sequence of claim1 or claim 2; b) constructing a vector comprising said isolatednucleotide sequence; c) transforming said vector into a host cell for atime and under conditions sufficient for expression of a Δ4-desaturaseencoded by said nucleotide sequence; d) exposing said expressedΔ4-desaturase to a polyunsaturated fatty acid substrate, in order toconvert said substrate to a product polyunsaturated fatty acid; and e)isolating said product polyunsaturated fatty acid.
 9. The methodaccording to claim 8, wherein said substrate is adrenic acid orω3-docosapentaenoic acid and said product polyunsaturated fatty acid isω6-docosapentaenoic acid or docosahexaenoic acid, respectively.
 10. Themethod according to claim 8 further comprising the step of exposing saidproduct polyunsaturated fatty acid to a desaturase in order to convertsaid product polyunsaturated fatty acid to another polyunsaturated fattyacid.
 11. The method according to claim 10 wherein said productpolyunsaturated fatty acid is ω6-docosapentaenoic acid and said anotherpolyunsaturated fatty acid is docosahexaenoic acid.